Tumor growth controlling method targeting galactosylceramide expression factor-1

ABSTRACT

The object of the present invention is to provide an anti-tumor agent having an anti-angiogenic effect and/or an anti-tumor growth effect or a method for tumor growth inhibition. 
     The present invention provides an anti-tumor agent characterized by having an anti-angiogenic effect and/or an anti-tumor growth effect, which contains a polypeptide of galactosylceramide expression factor-1 excluding the Q region, wherein the polypeptide comprises at least the C region or a part thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Phase, submitted pursuant to 35 U.S.C. §371,of International Patent Application No. PCT/JP2011/065139 filed on Jun.24, 2011, which claims priority to application no. JP 2010-144763 filedin Japan on Jun. 25, 2010. The disclosures of these prior applicationsare hereby incorporated by reference and in their entireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing is submitted herewith, pursuant to 37 C.F.R.1.821(c), as an ASCII compliant text file named “SeqList.txt”, which wascreated on Dec. 18, 2012 and has a size of 79 KB. The contents of theaforementioned “SeqList.txt” file are hereby incorporated by referenceand in their entirety.

TECHNICAL FIELD

The present invention relates to an anti-tumor agent and a method fortumor growth inhibition, each of which targets galactosylceramideexpression factor-1.

BACKGROUND ART

Galactosylceramide (GalCer) is the smallest glycosphingolipid composedof one galactose residue and one ceramide residue, which is highlyexpressed specifically in myelin oligodendrocytes and/or renalepithelial cells and has an insulating function and a high saltconcentration-resistant function.

Galactosylceramide expression factor-1 (GalCer expression factor-1(GEF-1)), which is an expression factor of galactosylceramide (Ogura,K., et al., J. Neurochem., 71, 1827-1836, 1998), is a protein composedof 4 regions, i.e., a zinc-finger sequence (FYVE)-containing region (Z),a proline-rich region (P), a coiled-coil sequence region (C), and aproline 2-rich/glutamine-rich region (O). Moreover, rat GEF-1 is a ratortholog of mouse Hrs (HGF-regulated tyrosine kinase substrate) (Komada,M., et al., Mol. Cell Biol., 15, 6213-6221, 1995) and is known to beinvolved in vesicular transport and intracellular signaling.

The inventors of the present invention have already found that GEF-1/Hrsinduces fibroblast-like morphological changes in MDCK cells, and havefurther found that GEF-1/Hrs is involved in the migration capacity ofcells (JP 2005-247735 A). However, GEF-1/Hrs and its Q region-deficientmutants showed no anti-tumor growth effect (JP 2005-247735 A).

On the other hand, tumors are known to release angiogenic factors (e.g.,VEGF and TGF-β) and thereby induce angiogenesis therein to compensatefor low oxygen and low nutrients during their growth process. Thus, astrategy to inhibit this tumor-induced angiogenesis has been designedfor inhibition of tumor growth. However, the relationship betweenGEF-1/Hrs and angiogenesis has not been known so far.

DISCLOSURE OF THE INVENTION

The present invention has been made under these circumstances, and theproblem to be solved by the present invention is to provide ananti-tumor agent having an anti-angiogenic effect and/or an anti-tumorgrowth effect or a method for tumor growth inhibition.

As a result of extensive and intensive efforts made to solve the aboveproblem, the inventors of the present invention have found that GEF-1 isinvolved in angiogenesis and tumor growth. Moreover, the inventors ofthe present invention have also found that inhibition of GEF-1expression allows inhibition of angiogenesis and tumor growth. Thesefindings led to the completion of the present invention.

Namely, the present invention is as follows.

(1) A tumor growth inhibitor, which contains a polypeptide ofgalactosylceramide expression factor-1 excluding the Q region, whereinthe polypeptide comprises at least the C region or a part thereof.(2) A tumor growth inhibitor, which comprises a polypeptide shown in (a)or (b) below:

(a) a polypeptide which consists of the amino acid sequence shown in SEQID NO: 8, 10 or 12; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in theamino acid sequence shown in SEQ ID NO: 8, 10 or 12 and which has ananti-tumor growth effect.

(3) A tumor growth inhibitor, which comprises a polypeptide shown in (a)or (b) below:

(a) a polypeptide which comprises at least 10 consecutive amino acidresidues in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in anamino acid sequence comprising at least 10 consecutive amino acidresidues in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12 andwhich has an anti-tumor growth effect.

(4) The tumor growth inhibitor according to (3) above, wherein thepolypeptide shown in (a) comprises an amino acid sequence shown in anyof SEQ ID NOs: 44 to 65.(5) A tumor growth inhibitor, which contains a polynucleotide encoding apolypeptide of galactosylceramide expression factor-1 excluding the Qregion, wherein the polypeptide comprises at least the C region or apart thereof.(6) A tumor growth inhibitor, which comprises a polynucleotide encodinga polypeptide shown in (a) or (b) below:

(a) a polypeptide which consists of the amino acid sequence shown in SEQID NO: 8, 10 or 12; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in theamino acid sequence shown in SEQ ID NO: 8, 10 or 12 and which has ananti-tumor growth effect.

(7) A tumor growth inhibitor, which comprises a polynucleotide encodinga polypeptide shown in (a) or (b) below:

(a) a polypeptide which comprises at least 10 consecutive amino acidresidues in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in anamino acid sequence comprising at least 10 consecutive amino acidresidues in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12 andwhich has an anti-tumor growth effect.

(8) A tumor growth inhibitor, which comprises a polynucleotide shown in(a) or (b) below:

(a) a polynucleotide which consists of the nucleotide sequence shown inSEQ ID NO: 7, 9 or 11; or

(b) a polynucleotide which is hybridizable under stringent conditionswith a polynucleotide consisting of a nucleotide sequence complementaryto the nucleotide sequence shown in SEQ ID NO: 7, 9 or 11 and whichencodes a polypeptide having an anti-tumor growth effect.

(9) A tumor growth inhibitor, which contains an expression inhibitor ofgalactosylceramide expression factor-1.(10) The tumor growth inhibitor according to (9) above, wherein theexpression inhibitor of galactosylceramide expression factor-1 is siRNAor shRNA against a polynucleotide encoding galactosylceramide expressionfactor-1.(11) The tumor growth inhibitor according to (10) above, wherein thepolynucleotide encoding galactosylceramide expression factor-1 comprisesa nucleotide sequence encoding a polypeptide shown in (a) or (b) below:

(a) a polypeptide which consists of the amino acid sequence shown in SEQID NO: 2, 4 or 6; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in theamino acid sequence shown in SEQ ID NO: 2, 4 or 6 and which has a tumorgrowth effect.

(12) The tumor growth inhibitor according to (10) above, wherein thepolynucleotide encoding galactosylceramide expression factor-1 comprisesa polynucleotide shown in (a) or (b) below:

(a) a polynucleotide which consists of the nucleotide sequence shown inSEQ ID NO: 1, 3 or 5; or

(b) a polynucleotide which is hybridizable under stringent conditionswith a polynucleotide consisting of a nucleotide sequence complementaryto the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 and whichencodes a polypeptide having a tumor growth effect.

(13) An angiogenesis inhibitor, which contains a polypeptide ofgalactosylceramide expression factor-1 excluding the Q region, whereinthe polypeptide comprises at least the C region or a part thereof (14)An angiogenesis inhibitor, which comprises a polypeptide shown in (a) or(b) below:

(a) a polypeptide which consists of the amino acid sequence shown in SEQID NO: 8, 10 or 12; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in theamino acid sequence shown in SEQ ID NO: 8, 10 or 12 and which has ananti-angiogenic effect.

(15) An angiogenesis inhibitor, which comprises a polypeptide shown in(a) or (b) below:

(a) a polypeptide which comprises at least 10 consecutive amino acidresidues in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in anamino acid sequence comprising at least 10 consecutive amino acidresidues in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12 andwhich has an anti-angiogenic effect.

(16) The angiogenesis inhibitor according to (15) above, wherein thepolypeptide shown in (a) comprises an amino acid sequence shown in anyof SEQ ID NOs: 44 to 65.(17) An angiogenesis inhibitor, which contains a polynucleotide encodinga polypeptide of galactosylceramide expression factor-1 excluding the Qregion, wherein the polypeptide comprises at least the C region or apart thereof (18) An angiogenesis inhibitor, which comprises apolynucleotide encoding a polypeptide shown in (a) or (b) below:

(a) a polypeptide which consists of the amino acid sequence shown in SEQID NO: 8, 10 or 12; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in theamino acid sequence shown in SEQ ID NO: 8, 10 or 12 and which has ananti-angiogenic effect.

(19) An angiogenesis inhibitor, which comprises a polynucleotideencoding a polypeptide shown in (a) or (b) below:

(a) a polypeptide which comprises at least 10 consecutive amino acidresidues in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in anamino acid sequence comprising at least 10 consecutive amino acidresidues in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12 andwhich has an anti-angiogenic effect.

(20) An angiogenesis inhibitor, which comprises a polynucleotide shownin (a) or

(b) below:

(a) a polynucleotide which consists of the nucleotide sequence shown inSEQ ID NO: 7, 9 or 11; or

(b) a polynucleotide which is hybridizable under stringent conditionswith a polynucleotide consisting of a nucleotide sequence complementaryto the nucleotide sequence shown in SEQ ID NO: 7, 9 or 11 and whichencodes a polypeptide having an anti-angiogenic effect.

(21) An angiogenesis inhibitor, which contains an expression inhibitorof galactosylceramide expression factor-1.(22) The angiogenesis inhibitor according to (21) above, wherein theexpression inhibitor of galactosylceramide expression factor-1 is siRNAor shRNA against a polynucleotide encoding galactosylceramide expressionfactor-1.(23) The angiogenesis inhibitor according to (22) above, wherein thepolynucleotide encoding galactosylceramide expression factor-1 comprisesa nucleotide sequence encoding a polypeptide shown in (a) or (b) below:

(a) a polypeptide which consists of the amino acid sequence shown in SEQID NO: 2, 4 or 6; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in theamino acid sequence shown in SEQ ID NO: 2, 4 or 6 and which has anangiogenic effect.

(24) The angiogenesis inhibitor according to (22) above, wherein thepolynucleotide encoding galactosylceramide expression factor-1 comprisesa polynucleotide shown in (a) or (b) below:

(a) a polynucleotide which consists of the nucleotide sequence shown inSEQ ID NO: 1, 3 or 5; or

(b) a polynucleotide which is hybridizable under stringent conditionswith a polynucleotide consisting of a nucleotide sequence complementaryto the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 and whichencodes a polypeptide having an angiogenic effect.

(25) An eliminator of cancer cell characteristics, which comprises apolypeptide shown in (a) or (b) below:

(a) a polypeptide which consists of the amino acid sequence shown in SEQID NO: 8, 10 or 12; or

(b) a polypeptide which consists of an amino acid sequence withdeletion, substitution or addition of one or several amino acids in theamino acid sequence shown in SEQ ID NO: 8, 10 or 12 and which has anelimination effect on cancer cell characteristics.

(26) The eliminator of cancer cell characteristics according to (25)above, which allows cancer cells to acquire the ability of contactinhibition.(27) A method for tumor growth inhibition, which comprises inhibitingthe expression of galactosylceramide expression factor-1.(28) A method for angiogenesis inhibition, which comprises inhibitingthe expression of galactosylceramide expression factor-1.

The present invention enables the provision of an anti-tumor agent whichcontains a polypeptide of galactosylceramide expression factor-1excluding the Q region, wherein the polypeptide comprises at least the Cregion or a part thereof. The present invention also enables theprovision of an anti-tumor agent which contains an expression inhibitorof galactosylceramide expression factor-1. The present invention is veryuseful as an anti-tumor agent because of having not only ananti-metastatic effect on cancer, but also an anti-angiogenic effectand/or an anti-tumor growth effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing the structure of GEF-1.

FIG. 2A shows the colony morphology of B16 cells, B16/bsr cells,B16/GEF-1 cells, B16/C cells and B16/shRNA cells.

FIG. 2B shows images of lung metastasis at 21 days after administrationof B16 cells, B16/GEF-1 cells, B16/C cells and B16/shRNA cells. In thefigure, “HE” denotes HE-stained images.

FIG. 2C shows the longevity (survival days) of mice after administrationof B16 cells, B16/bsr cells, B16/GEF-1 cells, B16/C cells and B16/shRNAcells.

FIG. 3 shows the results of tube formation test. A) Results of tubeformation obtained for KOP cells and KOP/C cells. B) Results of tubeformation obtained for MDCK/GEF-1 cells, MDCK cells and MDCK/C cells.

FIG. 4 shows the results of angiogenesis induction and inhibition underthe dorsal skin of mice (in vivo). The upper panel shows the conditionof angiogenesis, while the lower panel shows the results measured forvascular length and vascular area.

FIG. 5 shows the results measured for in vitro growth ability of B16/Ccells.

FIG. 6 shows the results measured for cell density and cell cycle ofB16/C cells.

FIG. 7 is a schematic view showing the structure of vectors used formeasurement of transcription factor activity and promoter activity.

FIG. 8 shows the results measured for effects of HGF and TGF-β on SMADtranscriptional activity in MDCK/GEF-1 cells and MDCK/C cells.

FIG. 9 shows the results measured for activity of various transcriptionfactors in MDCK/GEF-1 cells and MDCK/C cells.

FIG. 10 shows the results measured for activity of various transcriptionfactors in B16/GEF-1 cells, B16/shRNA cells and B16/C cells.

FIG. 11 shows the results measured for TGF-β-SMAD signal-induced PAI-1promoter activity in B16/bsr cells, B16/GEF-1 cells and B16/C cells.

FIG. 12 shows the results measured for changes in TGF-β levels containedin the medium.

FIG. 13A shows the results measured for growth ability of B16/GEF-1cells and B16/C cells in soft agar medium. The upper panel shows thecondition of colony formation, while the lower panel shows the resultsmeasured for relative values of colony counts.

FIG. 13B shows the results measured for growth ability of LLC/C cells insoft agar medium. The upper panel shows the condition of colonyformation, while the lower panel shows the results measured for relativevalues of colony counts.

FIG. 13C shows the growth morphology of MDCK cells and MDCK/GEF-1 cellsin soft agar medium.

FIG. 14A shows the results measured for tumorigenicity/tumor growthability of B16/GEF-1 cells, B16/shRNA cells and B16/C cells in C57BL/6mice.

FIG. 14B shows the results measured for tumorigenicity/tumor growthability of MDCK cells and MDCK/GEF-1 cells in nude mice.

FIG. 15 shows the results tested for inhibitory effects of GEF-1 siRNAson tube formation. The upper panel shows the condition of tube formationin siRNA-transfected cells, while the lower panel shows the resultsmeasured for relative values of % tube formation.

FIG. 16 shows the results tested for in vivo anti-tumor growth effect ofGEF-1 siRNA #3. The left panel shows the results measured for tumorvolume and tumor weight, while the right panel shows the appearance oftumors formed in nude mice.

FIG. 17 is a schematic view of GEF-1/C constituent oligopeptides.

FIG. 18A shows the results measured for effects of GEF-1/C constituentoligopeptides on SMAD transcriptional activity in B16 cells.

FIG. 18B shows the results measured for effects of GEF-1/C constituentoligopeptides on SMAD transcriptional activity in COLO205 cells.

FIG. 19 shows the results measured for effects of GEF-1/C constituentoligopeptides on tube formation in KOP cells.

FIG. 20A shows the results measured for effects of GEF-1/C constituentoligopeptides on B16 cell growth in soft agar medium.

FIG. 20B shows the results measured for effects of GEF-1/C constituentoligopeptides on HeLa cell growth in soft agar medium.

FIG. 20C shows the results measured for effects of GEF-1/C constituentoligopeptides on COLO205 cell growth in soft agar medium.

FIG. 20D shows the results measured for effects of GEF-1/C constituentoligopeptides on KATOIII cell growth in soft agar medium.

FIG. 21A shows the results measured for effects of GEF-1/C constituentoligopeptides on A549 cell growth in soft agar medium.

FIG. 21B shows the results measured for effects of GEF-1/C constituentoligopeptides on PT45 cell growth in soft agar medium.

FIG. 22 shows the results tested for in vivo anti-tumor growth effectsof GEF-1/C constituent oligopeptides using B16 cells. The left panelshows the results measured for tumor volume and tumor weight, while theright panel shows the appearance of tumors formed in nude mice.

FIG. 23 shows the results tested for in vivo anti-tumor growth effectsof GEF-1/C constituent oligopeptides using COLO205 cells. The left panelshows the results measured for tumor volume and tumor weight, while theright panel shows the appearance of tumors formed in nude mice.

FIG. 24 shows the results tested for in vivo anti-tumor growth effectsof GEF-1/C constituent oligopeptides using A549 cells. The left panelshows the results measured for tumor volume and tumor weight, while theright panel shows the appearance of tumors formed in nude mice.

FIG. 25 shows the results measured for in vitro growth ability of EL4/Ccells.

FIG. 26 shows the results measured for growth ability of PT45/C cells insoft agar medium. The upper panel shows the condition of colonyformation, while the lower panel shows the results measured for relativevalues of colony counts.

FIG. 27 shows the results measured for growth ability of COLO205/C cellsin soft agar medium. The upper panel shows the condition of colonyformation, while the lower panel shows the results measured for relativevalues of colony counts.

FIG. 28 shows the results tested for in vivo anti-tumor growth effect ofGEF-1/C constituent oligopeptide (r9-OP10-11) linked to a cell membranepermeable peptide using COLO205 cells. The left panel shows the resultsmeasured for changes in tumor volume and wet tumor weight, while theright panel shows the appearance of tumors formed in nude mice.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below. Thefollowing embodiments are illustrated to describe the present invention,and it is not intended to limit the present invention only to theseembodiments. The present invention can be implemented in various modeswithout departing from the spirit of the present invention. Moreover,this specification incorporates the contents disclosed in thespecification and drawings of Japanese Patent Application No.2010-144763 (filed on Jun. 25, 2010), based on which the presentapplication claims priority.

1. Summary

Galactosylceramide expression factor-1 (GalCer expression factor-1(GEF-1)) is a factor capable of inducing galactosylceramide expression(Ogura, K., et al., J. Neurochem., 71, 1827-1836, 1998). The inventorsof the present invention have already succeeded in inhibiting themetastatic ability of cancer cells by causing the cancer cells toexpress a polypeptide which comprises the C region of GEF-1 and is freefrom the Q region (JP 2005-247735 A). On the other hand, thispolypeptide was found not to significantly inhibit tumor growth (ibid).

However, the inventors of the present invention have studied again theeffect of GEF-1 on tumor growth, and hence have found that GEF-1enhances tumor growth. The inventors of the present invention havefurther studied the effect of GEF-1 on angiogenesis, and hence havefound that GEF-1 also enhances angiogenesis. Based on these findings,the C region of GEF-1 was then studied for its effects. As a result, ithas been found that tumor growth and angiogenesis can be inhibited whenthe C region of GEF-1 or a constituent oligopeptide thereof is allowedto express itself. Moreover, it has been found that tumor growth andangiogenesis can also be inhibited when GEF-1 expression is inhibited byusing a GEF-1 expression inhibitor. These results have indicated that apolypeptide covering the C region of GEF-1, a constituent oligopeptidethereof and a GEF-1 expression inhibitor have all of the three effects,i.e., cancer cell metastasis inhibition, angiogenesis inhibition andtumor growth inhibition, and are very useful as anti-tumor agents.Moreover, it has also been indicated that the anti-tumor agent of thepresent invention not only indirectly inhibits metastasis of cancercells and tumor growth through inhibition of tumor-induced angiogenesis,but also directly inhibits cancer metastasis through inhibition of themigration capacity of cancer cells per se, and further directly inhibitstumor growth through inhibition of the expression of transcriptionfactors involved in tumor growth of cancer cells. Thus, the anti-tumoragent of the present invention can exert the above three effects in asynergistic manner and hence is very useful for cancer or tumortreatment.

2. Galactosylceramide Expression Factor-1 (1) Functions ofGalactosylceramide Expression Factor-1

GEF-1 has the function of inducing high level expression of GalCer andits sulfated derivative sulfatide in MDCK cells derived from epithelialcells. In addition, GEF-1 has the function of inducingepithelial-mesenchymal transition (EMT) in MDCK cells to convert theMDCK cells into a fibroblast-like morphology. Epithelial-mesenchymaltransition (EMT) is an essential process in the early stage of embryonicmorphogenesis induced by signals such as Wnt, and is also greatlyinvolved in invasion and intravascular migration during cancer cellmetastasis (Bean, A., et al., Nature, 385, 826-829, 1997). In fact,GEF-1 has been shown to promote metastasis of cancer cells whenexpressed in mouse melanoma B16 cells, which are cancer cells. Moreover,GEF-1 also has an angiogenic effect and a tumor growth effect.

(2) GEF-1 Polypeptide

The GEF-1 polypeptide to be used in the present invention is not limitedin any way and may be of rat, human or mouse origin. Rat GEF-1 is ahomolog of mouse or human HGF-regulated tyrosine kinase substrate(hereinafter referred to as “Hrs” or “Hgs”). Amino acid sequencehomologies between Rat GEF-1 and mouse Hrs, between rat GEF-1 and humanHrs, and between mouse Hrs and human Hrs are as follows:

(a) rat GEF-1 and mouse Hrs have an amino acid sequence homology of 97%;

(b) rat GEF-1 and human Hrs have an amino acid sequence homology of 93%;and

(c) mouse Hrs and human Hrs have an amino acid sequence homology of 93%.

Thus, the GEF-1 polypeptide to be used in the present inventionencompasses a GEF-1 polypeptide of rat origin, an Hrs polypeptide ofhuman origin, and an Hrs polypeptide of mouse origin.

The amino acid sequences of GEF-1 (Hrs) polypeptides of rat, human andmouse origin are shown in SEQ ID NOs: 2, 4 and 6, respectively.Likewise, the nucleotide sequences of polynucleotides encoding GEF-1 ofrat, human and mouse origin are shown in SEQ ID NOs: 1, 3 and 5,respectively. The above nucleotide sequences and amino acid sequencesare registered in GenBank. GenBank accession numbers of the respectiveGEF-1 (Hrs) nucleotide sequences and amino acid sequences are shownbelow.

Rat GEF-1 (SEQ ID NOs: 1 and 2): AB002811

Human Hrs (SEQ ID NOs: 3 and 4): U43895

Mouse Hrs (SEQ ID NOs: 5 and 6): D50050

The GEF-1 polypeptide to be used in the present invention encompassesnot only the above polypeptide which consists of the amino acid sequenceshown in SEQ ID NO: 2, 4 or 6, but also a polypeptide which consists ofan amino acid sequence mutated by deletion, substitution or addition (orany combination thereof) of one or several amino acids in the amino acidsequence shown in SEQ ID NO: 2, 4 or 6 and which has an angiogeniceffect or a tumor growth effect.

Examples of the above amino acid sequence mutated by deletion,substitution or addition (or any combination thereof) of one or severalamino acids in the amino acid sequence shown in SEQ ID NO: 2, 4 or 6include:

(i) an amino acid sequence with deletion of 1 to 10 (e.g., 1 to 5,preferably 1 to 3, more preferably 1 to 2, even more preferably 1) aminoacids from the amino acid sequence shown in SEQ ID NO: 2, 4 or 6;

(ii) an amino acid sequence with substitution of other amino acids for 1to 10 (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, evenmore preferably 1) amino acids in the amino acid sequence shown in SEQID NO: 2, 4 or 6;

(iii) an amino acid sequence with addition of 1 to 10 (e.g., 1 to 5,preferably 1 to 3, more preferably 1 to 2, even more preferably 1) aminoacids to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6; and

(iv) an amino acid sequence mutated by any combination of (i) to (iii)above.

In the context of the present invention, the term “angiogenic effect” isintended to mean the effect of inducing in vitro tube formation or theeffect of inducing in vivo angiogenesis. Likewise, the term “tumorgrowth effect” is intended to mean the effect of promoting the growth ofcancer cells.

Moreover, “having an angiogenic effect or a tumor growth effect” isintended to mean having 10% or more, 20% or more, 30% or more, 40% ormore, 50% or more, 60% or more, 70% or more, 80% or more, preferably 90%or more activity, when compared to the angiogenic effect or tumor growtheffect of a polypeptide having the amino acid sequence shown in SEQ IDNO: 2, 4 or 6, which is set to 100.

The angiogenic effect may be confirmed in a known manner. For example,MDCK cells are caused to express a mutated polypeptide and tested toexamine whether their tube formation ability is enhanced or not, incomparison with control MDCK cells. Alternatively, the angiogenic effectmay be measured by determining the vascular length or vascular area inan angiogenesis induction test conducted under the dorsal skin of mice(DAS assay). On the other hand, the tumor growth effect may be measuredby causing cancer cells of any type to express a mutated polypeptide andmeasuring the amount of tumor growth using known procedures.

To prepare the above mutation-carrying polypeptides, mutations may beintroduced into polynucleotides by using a kit for mutation introductionbased on site-directed mutagenesis (e.g., Kunkel method or Gapped duplexmethod), as exemplified by a QuikChange™ Site-Directed Mutagenesis Kit(Stratagene), GeneTailor™ Site-Directed Mutagenesis Systems(Invitrogen), and TaKaRa Site-Directed Mutagenesis Systems (e.g.,Mutan-K, Mutan-Super Express Km; Takara Bio Inc., Japan). Alternatively,it is also possible to use techniques for site-directed mutagenesis asdescribed in “Molecular Cloning, A Laboratory Manual 2nd ed.” (ColdSpring Harbor Press (1989)), “Current Protocols in Molecular Biology”(John Wiley & Sons (1987-1997)), Kunkel (1985) Proc. Natl. Acad. Sci.USA 82: 488-92, Kramer and Fritz (1987) Method. Enzymol. 154: 350-67,Kunkel (1988) Method. Enzymol. 85: 2763-6, etc.

Furthermore, the GEF-1 polypeptide to be used in the present inventionencompasses not only those having the amino acid sequence shown in SEQID NO: 2, 4 or 6, but also those having amino acid sequences sharing ahomology of about 85% or more, preferably about 90% or more, morepreferably about 93% or more with the amino acid sequence shown in SEQID NO: 2, 4 or 6 and having an angiogenic effect or a tumor growtheffect (i.e., amino acid sequences substantially equivalent to the aminoacid sequence shown in SEQ ID NO: 2, 4 or 6). For homologydetermination, it is possible to use homology search tools such asFASTA, BLAST, PSI-BLAST and so on in a homology search site on theInternet (e.g., DNA Data Bank of Japan (DDBJ)). Alternatively, it ispossible to conduct a BLAST search in the National Center forBiotechnology Information (NCBI).

(3) Polynucleotide Encoding GEF-1

As described above, rat GEF-1 is a homolog of mouse or human Hrs. Thus,the polynucleotide encoding GEF-1 of the present invention encompassespolynucleotides encoding Hrs, e.g., those encoding human and mouse Hrs(SEQ ID NOs: 3 and 5), as well as polynucleotides encoding human andmouse Hrs polypeptides consisting of the amino acid sequences shown inSEQ ID NOs: 4 and 6, respectively. Moreover, a polynucleotide encodingrat Hrs is also included.

In the present invention, the polynucleotide encoding GEF-1 is notlimited in any way, as long as it is a polynucleotide comprising thenucleotide sequence shown in SEQ ID NO: 1, 3 or 5 or a nucleotidesequence encoding any of the above GEF-1 polypeptides. For example, inthe present invention, it is possible to use not only a polynucleotideencoding a polypeptide which consists of the amino acid sequence shownin SEQ ID NO: 2, 4 or 6, but also a polynucleotide encoding a mutatedpolypeptide which consists of an amino acid sequence with deletion,insertion, substitution or addition of one or several amino acids in theamino acid sequence shown in SEQ ID NO: 2, 4 or 6 and which has anangiogenic effect or a tumor growth effect.

As used herein, the term “polynucleotide” refers to a polymer composedof a plurality of bases or base pairs such as deoxyribonucleic acids(DNA) or ribonucleic acids (RNA), and encompasses DNA, cDNA, genomicDNA, chemically synthesized DNA and RNA. In addition, polynucleotidesoptionally containing unnatural artificial bases are also included.

The polynucleotide encoding GEF-1 of the present invention encompasses apolynucleotide which consists of the nucleotide sequence shown in SEQ IDNO: 1, 3 or 5, or a polynucleotide which is hybridizable under stringentconditions with a polynucleotide consisting of a sequence complementaryto the nucleotide sequence and which encodes a polypeptide having anangiogenic effect or a tumor growth effect. Such a polynucleotide may beobtained from human cDNA and genomic libraries by known hybridizationtechniques (e.g., colony hybridization, plaque hybridization, Southernblotting) using the polynucleotide which consists of the nucleotidesequence shown in SEQ ID NO: 1, 3 or 5 or a fragment thereof as a probe.For preparation of cDNA libraries, reference may be made to “MolecularCloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)).Alternatively, commercially available cDNA and genomic libraries mayalso be used for this purpose.

Hybridization conditions in the present invention include, for example,“2×SSC, 0.1% SDS, 25° C.,” “1×SSC, 0.1% SDS, 25° C.” or “0.2×SSC, 0.1%SDS, 42° C.” for stringent conditions, and “0.1×SSC, 0.1% SDS, 68° C.”for more stringent conditions. Those skilled in the art would be able todetermine the conditions required for obtaining the polynucleotideencoding the GEF-1 gene of the present invention in consideration of notonly these conditions including buffer salt concentration andtemperature, but also other various conditions including probeconcentration, probe length, reaction time and so on.

As to detailed procedures for hybridization, reference may be made to“Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring HarborPress (1989); particularly Section 9.47-9.58), “Current Protocols inMolecular Biology” (John Wiley & Sons (1987-1997); particularly Section6.3-6.4), etc.

The nucleotide sequence of the polynucleotide of the present inventioncan be confirmed by conventional sequencing techniques. For example,dideoxynucleotide chain termination sequencing (Sanger et al. (1977)Proc. Natl. Acad. Sci. USA 74: 5463) may be used for this purpose.Alternatively, an appropriate DNA sequencer (e.g., ABI PRISM (AppliedBiosystems)) may be used for sequence analysis.

3. C Region and Q Region of GEF-1 (1) Functions of the C Region and QRegion of GEF-1

As shown in FIG. 1, the GEF-1 protein is composed of 4 regions(domains), i.e., a zinc-finger sequence (FYVE)-containing region (Z), aproline-rich region (P), a coiled-coil sequence region (C) and a prolinerich-2/glutamine-rich region (Q) in this order from the amino-terminalend.

Polypeptides comprising at least the C-Q region of GEF-1 have theability to induce EMT, while polypeptides free from the Q region andcomprising the C region (e.g., ZPC, PC, C) have inhibitory activityagainst EMT induction. Moreover, in an in vitro cell migrationexperiment using a transwell chamber, cells expressing the full-lengthGEF-1 (ZPCQ) enhance their migration capacity, whereas GEF-1 mutantslacking the Q region and comprising the C region (ZPC, PC, C) cause asignificant decrease in their migration capacity, and in particular,cells expressing the C region show a remarkably significant decrease intheir migration capacity (JP 2005-247735 A).

Furthermore, as described later in the Example section, polypeptidescovering the C region of GEF-1 inhibit metastasis of cancer cells.

Thus, polypeptides of GEF-1 excluding the Q region, which comprise atleast the C region, i.e., polypeptides covering the ZPC region of GEF-1,preferably the PC region, more preferably the C region have ananti-metastatic effect on cancer cells. Moreover, polypeptides coveringthe C region of GEF-1 have an anti-angiogenic effect and an anti-tumorgrowth effect.

(2) Polypeptides of GEF-1 Excluding the Q Region, which Comprise atLeast the C Region

In the context of the present invention, the “polypeptide ofgalactosylceramide expression factor-1 excluding the Q region, whichcomprises at least the C region” is intended to mean a polypeptide whichis free from the Q region of GEF-1 and comprises the C region of GEF-1.Examples of such a polypeptide include a polypeptide covering the Cregion of GEF-1 (GEF-1/C), as well as a polypeptide comprising thefull-length P region or a part thereof at the amino-terminal side of theC region (GEF-1/PC) and a polypeptide comprising the full-length Zregion or a part thereof at the amino-terminal side of the PC region(GEF-1/ZPC), with the polypeptide covering the C region (GEF-1/C) beingpreferred.

The polypeptides GEF-1/ZPC, GEF-1/PC and GEF-1/C and mutants thereof canbe readily prepared by those skilled in the art on the basis of Table 1shown below and the mutant preparation techniques mentioned above. Thepositions of the Z, P, C and Q regions in the amino acid sequences shownin SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 are shown in Table 1below.

TABLE 1 Positions of Z, P, C and Q regions in the full-length amino acidsequence of GEF-1/Hrs Z P C Q Rat 1 to 233 234 to 390 391 to 562 563 to771 (SEQ ID NO: 2) Human 1 to 233 234 to 390 391 to 563 564 to 777 (SEQID NO: 4) Mouse 1 to 233 234 to 390 391 to 561 562 to 775 (SEQ ID NO: 6)(3) Polynucleotides Encoding Polypeptides of GEF-1 Excluding the QRegion, which Comprise at Least the C Region

Examples of polynucleotides encoding the polypeptides described in (2)above include polynucleotides encoding GEF-1/C, GEF-1/PC and GEF-1/ZPCshown above. These polynucleotides and mutants thereof can be readilyprepared by those skilled in the art on the basis of Table 2 shown belowand the hybridization techniques mentioned above. The positions of theZ, P, C and Q regions in the nucleotide sequences of SEQ ID NO: 1 (ratGEF-1), SEQ ID NO: 3 (human Hrs) and SEQ ID NO: 5 (mouse Hrs) are shownin Table 2 below.

TABLE 2 Positions of polynucleotides encoding Z, P, C and Q regions Z PC Q Rat 21 to 719 720 to 1190 1191 to 1706 1707 to 2333 (SEQ ID NO: 1)Human 76 to 774 775 to 1245 1246 to 1764 1765 to 2406 (SEQ ID NO: 3)Mouse 37 to 735 736 to 1206 1207 to 1719 1720 to 2361 (SEQ ID NO: 5)

4. Polypeptide Covering the C Region of GEF-1 (1) Polypeptide Coveringthe C Region of GEF-1 (GEF-1/C)

In the context of the present invention, the polypeptide of GEF-1excluding the Q region, which comprises at least the C region isintended to include a polypeptide covering the C region of GEF-1.

The polypeptide covering the C region of GEF-1 has an anti-metastaticeffect on cancer cells, an anti-angiogenic effect and an anti-tumorgrowth effect.

The polypeptide covering the C region of GEF-1 includes a polypeptidewhich consists of the amino acid sequence shown in SEQ ID NO: 8 (ratGEF-1/C), SEQ ID NO: 10 (human Hrs/C) or SEQ ID NO: 12 (mouse Hrs/C).

In addition to the above polypeptide which consists of the amino acidsequence shown in SEQ ID NO: 8, 10 or 12, other members of thepolypeptide covering the C region of GEF-1 include a polypeptide whichconsists of an amino acid sequence mutated by deletion, substitution oraddition (or any combination thereof) of one or several amino acids inthe amino acid sequence shown in SEQ ID NO: 8, 10 or 12 and which has ananti-angiogenic effect or an anti-tumor growth effect.

Examples of the above amino acid sequence mutated by deletion,substitution or addition (or any combination thereof) of one or severalamino acids in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12include:

(i) an amino acid sequence with deletion of 1 to 10 (e.g., 1 to 5,preferably 1 to 3, more preferably 1 to 2, even more preferably 1) aminoacids from the amino acid sequence shown in SEQ ID NO: 8, 10 or 12;

(ii) an amino acid sequence with substitution of other amino acids for 1to 10 (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, evenmore preferably 1) amino acids in the amino acid sequence shown in SEQID NO: 8, 10 or 12;

(iii) an amino acid sequence with addition of 1 to 10 (e.g., 1 to 5,preferably 1 to 3, more preferably 1 to 2, even more preferably 1) aminoacids to the amino acid sequence shown in SEQ ID NO: 8, 10 or 12; and

(iv) an amino acid sequence mutated by any combination of (i) to (iii)above.

The anti-angiogenic effect and anti-tumor growth effect in the contextof the present invention have the same definitions and may be measuredin the same manner as described above. Likewise, to prepare the abovemutation-carrying polypeptides, mutations may be introduced intopolynucleotides in the same manner as described above.

The polypeptide covering the C region of GEF-1 encompasses not onlythose having the amino acid sequence shown in SEQ ID NO: 8, 10 or 12,but also those having amino acid sequences sharing a homology of about85% or more, preferably about 90% or more, more preferably about 95% ormore with the amino acid sequence shown in SEQ ID NO: 8, 10 or 12 andhaving an anti-angiogenic effect or an anti-tumor growth effect (i.e.,amino acid sequences substantially equivalent to the amino acid sequenceshown in SEQ ID NO: 8, 10 or 12). Homology search may be accomplished inthe same manner as described above.

(2) Polynucleotide Encoding the Polypeptide Covering the C Region ofGEF-1 (GEF-1/C)

In the present invention, the polynucleotide encoding GEF-1/C is notlimited in any way, as long as it is a polynucleotide comprising thenucleotide sequence shown in SEQ ID NO: 7, 9 or 11 or a nucleotidesequence encoding the above polypeptide covering the C region of GEF-1.For example, in the present invention, it is possible to use not only apolynucleotide encoding a polypeptide which consists of the amino acidsequence shown in SEQ ID NO: 8, 10 or 12, but also a polynucleotideencoding a mutated polypeptide which consists of an amino acid sequencewith deletion, insertion, substitution or addition of one or severalamino acids in the amino acid sequence shown in SEQ ID NO: 8, 10 or 12and which has an anti-angiogenic effect or an anti-tumor growth effect.

The polynucleotide encoding GEF-1/C of the present invention encompassesa polynucleotide which consists of the nucleotide sequence shown in SEQID NO: 7, 9 or 11, or a polynucleotide which is hybridizable understringent conditions with a polynucleotide consisting of a sequencecomplementary to the nucleotide sequence and which encodes a polypeptidehaving an anti-angiogenic effect or an anti-tumor growth effect. Such apolynucleotide may be obtained in a known manner on the basis of thepolynucleotide which consists of the nucleotide sequence shown in SEQ IDNO: 7, 9 or 11. Moreover, conditions required for the abovehybridization and techniques used for nucleotide sequencing are the sameas described above.

5. Constituent Oligopeptides Derived from the C Region of GEF-1 (GEF-1/CConstituent Oligopeptides)

In view of the fact that the polypeptide covering the C region of GEF-1has an anti-metastatic effect on cancer cells, an anti-angiogenic effectand an anti-tumor growth effect, partial polypeptides thereof may alsohave the same effects. As described later in the Example section,constituent oligopeptides derived from the C region of GEF-1 actuallyhave an anti-metastatic effect on cancer cells, an anti-angiogeniceffect and an anti-tumor growth effect.

Thus, the polypeptide to be used in the present invention encompassespolypeptides of GEF-1 excluding the Q region, which comprise at leastthe C region or a part thereof. As used herein, the phrase “a partthereof” is intended to mean a part of the C region, and its amino acidsequence may have any length. Moreover, oligopeptides composed of 2 to20 amino acid residues also fall within the “polypeptide” in the contextof the present invention.

Examples of polypeptides comprising a part of the C region includepolypeptides comprising at least 6 or more consecutive amino acidresidues, preferably 8 or more consecutive amino acid residues, morepreferably 10 or more consecutive amino acid residues in the amino acidsequence shown in SEQ ID NO: 8, 10 or 12. More specific examples includepolypeptides comprising 6 to 140 consecutive amino acid residues,preferably 8 to 30 consecutive amino acid residues, more preferably 8 to20 consecutive amino acid residues, even more preferably 10 consecutiveamino acid residues in the amino acid sequence shown in SEQ ID NO: 8, 10or 12.

The range of amino acid sequences selected as “a part of the C region”is not limited in any way, as long as the selected amino acid sequencesare within the amino acid sequence shown in SEQ ID NO: 8, 10 or 12. Forexample, in the case of the amino acid sequence shown in SEQ ID NO: 10,preferred is an amino acid sequence covering residues 33 (methionine) to172 (alanine), and more preferred is an amino acid sequence coveringresidues 83 (lysine) to 152 (glutamic acid). Likewise, in the case ofthe amino acid sequence shown in SEQ ID NO: 8 or 12, an amino acidsequence corresponding to these residues can also be selected as a partof the C region.

Further, amino acid sequences covering a part of the C region include,but are not limited to, those comprising any of the following amino acidsequences, by way of example:

(SEQ ID NO: 44) OP20-1: MKSNHMRGRSITNDSAVLSL (SEQ ID NO: 45) OP20-2:ITNDSAVLSLFQSINTMHPQ (SEQ ID NO: 46) OP20-3: FQSINTMHPQLLELLNQLDE(SEQ ID NO: 47) OP20-4: LLELLNQLDERRLYYEGLQD (SEQ ID NO: 48) OP20-5:RRLYYEGLQDKLAQIRDARG (SEQ ID NO: 49) OP20-6: KLAQIRDARGALSALREEHR(SEQ ID NO: 50) OP20-7: ALSALREEHREKLRRAAEEA (SEQ ID NO: 51) OP20-8:EKLRRAAEEAERQRQIQLAQ (SEQ ID NO: 52) OP20-9: ERQRQIQLAQKLEIMRQKKQ(SEQ ID NO: 53) OP20-10: KLEIMRQKKQEYLEVQRQLA (SEQ ID NO: 54) OP20-11:EYLEVQRQLAIQRLQEQEKE (SEQ ID NO: 55) OP20-12: IQRLQEQEKERQMRLEQQKQ(SEQ ID NO: 56) OP20-13: RQMRLEQQKQTVQMRAQMPA (SEQ ID NO: 57) OP10-1:MGRGSGTFER (SEQ ID NO: 58) OP10-3: FQSINTMHPQ (SEQ ID NO: 59) OP10-6:KLAQIRDARG (SEQ ID NO: 60) OP10-7: ALSALREEHR (SEQ ID NO: 61) OP10-8:EKLRRAAEEA (SEQ ID NO: 62) OP10-9: ERQRQIQLAQ (SEQ ID NO: 63) OP10-10:KLEIMRQKKQ (SEQ ID NO: 64) OP10-11: EYLEVQRQLA (SEQ ID NO: 65) OP10-12:IQRLQEQEKE

The above polypeptides comprising a part of the C region also encompassa polypeptide which consists of an amino acid sequence mutated bydeletion, substitution or addition (or any combination thereof) of oneor several amino acids in an amino acid sequence comprising at least 6to 10 consecutive amino acid residues in the amino acid sequence shownin SEQ ID NO: 8, 10 or 12 and which has an anti-angiogenic effect or ananti-tumor growth effect. As used herein, the term “several amino acids”is intended to mean 1 to 10 (e.g., 1 to 5, preferably 1 to 3, morepreferably 1 to 2, even more preferably 1) amino acids. When the totalnumber of amino acid residues contained in the above polypeptide isaround 20, the term is intended to mean 1 to 5 amino acids. When thetotal number of amino acid residues contained in the above polypeptideis around 10, the term is intended to mean 1 to 3 amino acids.

The above polypeptides comprising a part of the C region furtherencompass peptides consisting of an amino acid sequence covering a partof the C region, or mutants thereof, each having a cell membranepermeable peptide added (linked) thereto.

A cell membrane permeable peptide is a basic peptide rich in lysineresidues and arginine residues, and known examples include HIV-1virus-derived TAT protein (trans-activator of transcription protein) andso on. The percentage of basic amino acid residues contained in a cellmembrane permeable peptide is, for example, 30% to 100%, preferably100%. As an artificial synthetic cell membrane permeable peptide, a cellmembrane permeable peptide composed of 7 to 11 arginine residues linkedto each other is known. Moreover, arginine residues contained in such acell membrane permeable peptide are preferably in D-form.

A cell membrane permeable peptide may be linked to any substance, andthe substance carrying this peptide will be able to permeate the cellmembrane and migrate into cells.

In the present invention, for example, a cell membrane permeable peptidemay be linked to the above peptide consisting of an amino acid sequencecovering a part of the C region, whereby this peptide consisting of anamino acid sequence covering a part of the C region can be transferredinto target cells.

For example, a cell membrane permeable peptide comprising 9 residues ofD-arginine (rrrrrrrrrGPG (SEQ ID NO: 66)) is linked to the above peptideconsisting of an amino acid sequence covering a part of the C region,OP10-11 (EYLEVQRQLA), to prepare a polypeptide (rrrrrrrrrGPGEYLEVQRQLA(SEQ ID NO: 67)), and this polypeptide is brought into contact withtarget cells, whereby OP10-11 can be introduced into the target cells.

The angiogenic effect and tumor growth effect of the polypeptidescomprising a part of the C region have the same definitions and may bemeasured in the same manner as described above. Likewise, to prepare theabove mutation-carrying polypeptides, mutations may be introduced intopolynucleotides in the same manner as described above.

Moreover, polynucleotides encoding the above polypeptides comprising apart of the C region can be readily prepared by those skilled in the artin accordance with known procedures on the basis of the amino acidsequence information of the above polypeptides comprising a part of theC region and/or the nucleotide sequence information of polynucleotidesencoding the above C region polypeptides, etc.

6. Preparation of Polypeptide

The polypeptide of GEF-1 excluding the Q region, which comprises atleast the C region or a part thereof (hereinafter referred to as “thepolypeptide of the present invention”) may be prepared by using knownprocedures. Detailed procedures are as follows.

(1) Preparation of Expression Vector

Any vector may be used to express the polypeptide of the presentinvention, as long as it can be held in a host cell where expression isto occur, and examples include plasmid DNAs, bacteriophages, etc.

Examples of plasmid DNAs include pME18S, pcDNA3, pBR322, pUC18, pUC19,pUC118, pUC119, pBluescript and so on, although other plasmids are alsopossible, such as those derived from E. coli, Bacillus subtilis, yeast,etc. Examples of phage DNAs include λphages (Charon4A, Charon21A, EMBL3,EMBL4, λgt10, λgt11) and so on.

For integration of a polynucleotide encoding the polypeptide of thepresent invention into a vector, procedures involving cleavage with anappropriate restriction enzyme and the subsequent ligation by treatmentwith ligase may be used for this purpose (see, e.g., Molecular Cloning,CSHL Press cited above).

For example, a polynucleotide encoding the polypeptide of the presentinvention may be integrated as follows: primers are designed for each ofthe Z, P and C regions to amplify their respective polynucleotides,these regions are each amplified by PCR or other techniques, and only adesired segment of each region is then ligated to a vector using arestriction enzyme or ligase. Alternatively, primers may be designed toamplify a target segment, and the target segment may be amplified byPCR, and ligated to a vector.

(2) Transformation

Any host may be used in the present invention, as long as it willexpress a polypeptide comprising the C region of GEF-1 and so on uponintroduction of a gene encoding the polypeptide comprising the C regionof GEF-1 and so on. Examples include, but are not limited to, mammaliancells, bacteria (e.g., bifidobacteria, lactic acid bacteria, E. coli),insect cells, yeast, fungi, etc.

Introduction of recombinant DNA into a host may be accomplished in aknown manner. Techniques used to introduce the above vector into a hostinclude, for example, calcium phosphate transfection, DEAE-dextrantransfection, electroporation, cationic lipid transfection, etc.

Moreover, confirmation as to whether DNA has been introduced may beaccomplished by using a selection marker gene (e.g., ampicillinresistance gene, neomycin resistance gene, hygromycin resistance gene,tetracycline resistance gene, chloramphenicol resistance gene, kanamycinresistance gene, zeocin resistance gene, blasticidin resistance gene).

(3) Production of Polypeptide

The polypeptide of the present invention can be obtained by culturingthe above transformant carrying a polynucleotide encoding thispolypeptide or a mutant thereof, and collecting the polypeptide from thecultured product.

The term “cultured product” is intended to mean a culture supernatant,cultured cells, cultured microorganisms, or a homogenate of cells ormicroorganisms. The transformant of the present invention may becultured in accordance with standard procedures commonly used for hostculture.

In the case of culturing a recombinant carrying an expression vector inwhich an inducible transcription promoter is used as a promoter, aninducer may optionally be added to the medium. When IPTG is used as aninducer, IPTG is added in an amount of 0.1 to 1.0 mM at 2 to 12 hoursafter the initiation of culture, and after IPTG addition, culture isfurther continued for 1 to 12 hours.

If the polypeptide of the present invention is accumulated within thecultured microorganisms or cells, a homogenizer or the like may be usedto homogenize the microorganisms or cells, thereby collecting thedesired polypeptide. If the polypeptide of the present invention isproduced outside microorganisms or cells, the cultured solution is useddirectly or further treated by centrifugation or the like to remove themicroorganisms or cells. Then, the above cultured solution is subjectedto ammonium sulfate precipitation or other operations to collect thepolypeptide, which is optionally further isolated and purified byvarious chromatographic techniques, etc.

Alternatively, in the present invention, a cell-free protein synthesissystem may be used to produce the polypeptide of the present invention,without using any living cells.

A cell-free protein synthesis system is a system for protein synthesisfrom a cell extract in an artificial container (e.g., a test tube). Forexample, such a system is configured to synthesize a protein on aribosome by reading the information of mRNA. It should be noted that thecell-free protein synthesis system to be used in the present inventionencompasses a cell-free transcription system in which RNA is synthesizedusing DNA as a template.

The above cell extract may be an extract derived from eukaryotic orprokaryotic cells, as exemplified by extracts of wheat germ, rabbitreticulocytes, mouse L-cells, HeLa cells, CHO cells, budding yeast, E.coli cells and so on. It should be noted that these cell extracts may ormay not be concentrated.

In the present invention, cell-free protein synthesis may beaccomplished by using a commercially available kit. Examples of such akit include reagent kits PROTEIOS™ (Toyobo Co., Ltd., Japan) and TNT™System (Promega), as well as synthesizers PG-Mate™ (Toyobo Co., Ltd.,Japan) and RTS (Roche Diagnostics), etc.

The polypeptide of the present invention obtained by cell-free proteinsynthesis can be purified by selecting an appropriate chromatographictechnique, as described above. Moreover, confirmation as to whether thepolypeptide of the present invention has been isolated and purified maybe accomplished by SDS-PAGE or other techniques.

Alternatively, the polypeptide of the present invention may also beobtained from the full-length GEF-1 or the like by cleavage withcyanogen bromide or a peptidase, etc. Any peptidase may be used for thispurpose as long as it is capable of cleaving the intended polypeptide,and examples include trypsin, chymotrypsin, lysyl endopeptidase and soon.

(4) Peptide Synthesis

The polypeptide of the present invention can be obtained by chemicalsynthesis. Peptide synthesis may be accomplished in a known manner usinga synthesizer such as a Model 433A peptide synthesizer (AppliedBiosystems) or PSSM-8 (Shimadzu Corporation, Japan). Alternatively, thepolypeptide of the present invention may also be obtained by beingpurchased from a company (e.g., Hayashi-Kasei Co., Ltd., Japan)entrusted with peptide synthesis. A polypeptide having a cell membranepermeable peptide linked thereto may also be obtained in the samemanner.

7. GEF-1 Expression Inhibitor (1) GEF-1 Expression Inhibitor

GEF-1 promotes cancer metastasis, induces angiogenesis and promotestumor growth. Thus, these effects can be inhibited when inhibiting theexpression of this GEF-1.

Examples of GEF-1 expression inhibitors include nucleic acids used forRNA interference (microRNA, shRNA, siRNA), antisense nucleic acids,decoy nucleic acids, or aptamers. These expression inhibitors arecapable of inhibiting GEF-1 expression. The nucleotide sequence of theGEF-1 gene to be inhibited is already known, as described above, andsequence information can be obtained for each case. In the presentinvention, the nucleotide sequence of the GEF-1 gene is as shown in SEQID NO: 1, 3 or 5, although not only a coding region of GEF-1, but also anon-coding region may be used as a target.

(2) RNA Interference

In the present invention, RNA interference (RNAi) against the GEF-1 genemay be used to inhibit the gene from being expressed. Examples ofnucleic acids used for such RNAi include siRNA, microRNA (miRNA) andshRNA.

siRNA (small interfering RNA) molecules are various RNAs correspondingto the target GEF-1 gene. Such RNAs include mRNA, post-transcriptionallymodified RNA of the GEF-1 gene, etc.

In general, a target sequence on mRNA may be selected from the cDNAsequence corresponding to the mRNA. However, the target sequence is notlimited to this region in the present invention.

siRNA molecules may be designed on the basis of the criteria well knownin the art. For example, as a target segment in the target mRNA, it ispossible to select a segment covering 15 to 30 consecutive bases,preferably 19 to 25 consecutive bases, preferably starting with AA, TA,GA or CA. siRNA molecules have a GC ratio of 30% to 70%, preferably 35%to 55%.

Because of forming double-stranded portions, siRNA can be produced as asingle-stranded hairpin RNA molecule folding on its own nucleic acid.Although siRNA molecules may be obtained by standard chemical synthesis,they can also be biologically produced using an expression vector.

For introduction of siRNA into cells, it is possible to use, e.g.,procedures in which synthesized siRNA is ligated to plasmid DNA and thenintroduced into cells, or procedures in which double-stranded RNA isannealed.

In the present invention, it is also possible to use commerciallyavailable siRNAs such as Stealth™ RNAi, Stealth™ Select RNAi(Invitrogen), etc.

In the present invention, shRNA may also be used for providing RNAieffect. shRNA is an RNA molecule called short hairpin RNA, which has astem loop structure because some single-stranded regions formcomplementary strands with other regions.

shRNA may be designed to form a stem loop structure as a part thereof.For example, assuming that a sequence covering a certain region isdesignated as sequence A, and a strand complementary to the sequence Ais designated as sequence B, shRNA is design to comprise the sequence A,a spacer and the sequence B linked in this order on a single RNA strandand to have an overall length of 45 to 60 bases. The spacer may alsohave any length. Although the sequence A is a sequence covering apartial region of the target GEF-1 gene, there is no particularlimitation on the target region and any region may be selected as acandidate for the target region. In addition, the sequence A has alength of 19 to 25 bases, preferably 19 to 21 bases.

The thus designed shRNA may be produced, for example, as follows: apolynucleotide serving as a template of shRNA is amplified by PCR andthen introduced into an appropriate vector for shRNA expression such aspSuperior.neo.gfp vector (Oligoengine), followed by gene transfer tointroduce the resulting shRNA expression vector into target cells.

Further, in the present invention, microRNA may be used to inhibit GEF-1expression. microRNA (miRNA) is an intracellular single-stranded RNAmolecule having a length of about 20 to 25 bases and is a kind of ncRNA(non-coding RNA) which is considered to have the function of regulatingthe expression of other genes. miRNA is generated through processingupon transcription into RNA and is present as a nucleic acid capable offorming a hairpin structure which inhibits the expression of a targetsequence.

Since miRNA is also a nucleic acid based on RNAi, miRNA may also bedesigned and synthesized in the same manner as in the case of shRNA orsiRNA.

(3) Antisense Nucleic Acid

In another embodiment of the present invention, an antisense nucleicacid may be used to inhibit GEF-1 expression. An antisense nucleic acidis a single-stranded nucleic acid sequence (either RNA or DNA) capableof binding to mRNA or DNA sequence of the GEF-1 gene. Such an antisensenucleic acid sequence has a length of at least 14 nucleotides,preferably 14 to 100 nucleotides. The antisense nucleic acid binds tothe above gene sequence to form a duplex and thereby inhibit thetranscription or translation of the GEF-1 gene.

The antisense nucleic acid may be prepared by chemical or biochemicalsynthesis procedures known in the art. For example, it is possible touse nucleic acid synthesis procedures using a commonly used DNAsynthesizer. The antisense nucleic acid is introduced into cancer cellsexpressing GEF-1, for example, by various gene transfer techniquesincluding DNA transfection or electroporation.

(4) Decoy Nucleic Acid

In yet another embodiment of the present invention, a nucleic acidserving as a decoy, which is called a decoy nucleic acid, may be used toinhibit GEF-1 expression.

A decoy nucleic acid is a nucleic acid that inhibits GEF-1 geneexpression through binding to the GEF-1 gene to inhibit promoteractivity, and is intended to mean a short nucleic acid serving as adecoy that comprises a binding site for a transcription factor. Whenthis nucleic acid is introduced into cells, a transcription factor willbind to this nucleic acid. As a result, the transcription factor will becompetitively inhibited from binding to its genomic binding site andthus inhibited from being expressed.

Examples of a decoy nucleic acid preferred in the present inventioninclude a nucleic acid binding to a promoter for the GEF-1 gene, or anucleic acid binding to a promoter for mRNA of GEF-1 or for a factorlocated upstream of GEF-1. Such a decoy nucleic acid may be designed asa single or double strand on the basis of the promoter sequence for theGEF-1 gene. Although the decoy nucleic acid may have any length, itslength is 15 to 60 bases, preferably 20 to 30 bases.

The decoy nucleic acid may be either DNA or RNA, and may also includemodified nucleic acids.

The decoy nucleic acid to be used in the present invention may beprepared by chemical or biochemical synthesis procedures known in theart. For example, it is possible to use nucleic acid synthesisprocedures using a DNA synthesizer commonly used in gene recombinationtechnology. Alternatively, a nucleotide sequence serving as a templatemay be isolated or synthesized, followed by PCR or gene amplificationusing a cloning vector. Further, to obtain a decoy nucleic acid which ismore stable within cells, bases and other constituents may be modifiedby chemical modifications such as alkylation, acylation, etc.

It should be noted that in the case of using a decoy nucleic acid,analysis of the promoter's transcriptional activity may be accomplishedby using standard assays such as luciferase assay, gel shift assay,RT-PCR, etc. Kits for performing these assays are also commerciallyavailable (e.g., a promega dual luciferase assay kit).

(5) Aptamer

Furthermore, in the present invention, an aptamer may be used to inhibitGEF-1 expression.

An aptamer is a synthetic DNA or RNA molecule or a peptidic molecule,which has the ability to bind specifically to a target substance, andcan be synthesized chemically in a test tube within a short time. Thus,an aptamer may be obtained on the basis of the nucleotide sequence shownin SEQ ID NO: 1, 3 or 5, or alternatively, may be obtained byevolutionary engineering procedures known as in vitro selection or SELEXtechniques.

A nucleic acid aptamer is rapidly degraded and removed in blood flow bythe action of nucleases, and hence it is preferred that the aptamer hasoptionally been subjected to molecular modification, e.g., with apolyethylene glycol (PEG) chain to thereby extend its half-life.

8. Anti-Tumor Agent

The polypeptide of the present invention, the vector expressing thispolypeptide, as well as shRNA and siRNA prepared in the presentinvention have an anti-metastatic effect on cancer, an anti-angiogeniceffect and/or an anti-tumor growth effect. Thus, compositions comprisingthem can be used as anti-tumor agents.

The anti-tumor agent of the present invention is capable of not onlyindirectly inhibiting cancer metastasis and tumor growth throughinhibition of tumor-induced angiogenesis, but also inhibiting cancermetastasis through inhibition of the migration capacity of cancer cells,and further inhibiting tumor growth through inhibition of the expressionof transcription factors involved in tumor growth. Thus, the anti-tumoragent of the present invention is very useful for cancer treatment whencompared to known anti-tumor agents, because it achieves directinhibition of three events, i.e., cancer metastasis, angiogenesis andtumor growth, and further allows tumor treatment as a result of asynergistic effect produced thereby.

In the context of the present invention, the term “cancer” or “tumor” isintended to include, but is not limited to, solid cancers such as braintumor, esophageal cancer, tongue cancer, lung cancer, breast cancer,pancreatic cancer, gastric cancer, small intestinal or duodenal cancer,colorectal cancer (colon cancer, rectal cancer), bladder cancer, kidneycancer, liver cancer, prostate cancer, uterine cancer, uterine cervicalcancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyngealcancer, sarcoma (e.g., osteosarcoma, chondrosarcoma, Kaposi's sarcoma,myosarcoma, angiosarcoma, fibrosarcoma), melanoma and so on, as well asblood tumors such as leukemia (e.g., chronic myelogenous leukemia (CML),acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) andacute lymphocytic leukemia (ALL)), lymphoma, multiple myeloma (MM) andso on.

In the context of the present invention, the term “angiogenesis” isintended to include both physiological angiogenesis and pathologicalangiogenesis, preferably refers to pathological angiogenesis.Physiological angiogenesis is observed at a limited site and during alimited period, and is inhibited under normal conditions. In contrast,pathological angiogenesis is not under the control of in vivo regulatorymechanisms and hence will occur disorderly, and is observed in cancer ortumor, diabetic retinopathy, rheumatoid arthritis, psoriasis vulgaris,atherosclerosis, etc.

In the context of the present invention, pathological angiogenesis ispreferably tumor-induced angiogenesis.

The term “metastasis” is intended to mean that cancer cells aretransported via the blood or lymphatic system or during surgicaloperation from their primary focus to another distant site where thecancer cells will form a new growth focus. Alternatively, the term“metastasis” refers to “the ability of cancer cells to propagatethemselves and form a new growth focus at a discrete site (i.e., theability to form metastasis)” (Hill, R. P, “Metastasis”, The BasicScience of Oncology, edited by Tannock, et. al., 178-195 (McGraw-Hill,New York, 1992)).

The anti-tumor agent of the present invention may comprise apharmaceutically acceptable carrier, in addition to the polypeptide ofthe present invention, the vector expressing this polypeptide, or shRNAor siRNA prepared in the present invention. The term “pharmaceuticallyacceptable carrier” refers to any type of carrier (e.g., liposomes,lipid vesicles, micelles), diluent, excipient, wetting agent, bufferingagent, suspending agent, lubricant, adjuvant, emulsifier, disintegrant,absorbent, preservative, surfactant, coloring agent, flavoring agent orsweetener, which is suitable for use as an anti-tumor agent.

The anti-tumor agent of the present invention may be formulated into anydosage form, such as injections, lyophilized preparations, tablets, hardcapsules, soft capsules, granules, powders, pills, syrups,suppositories, poultices, ointments, creams, eye drops, etc.

The anti-tumor agent of the present invention is topically orsystemically administered by any means known to those skilled in theart. The dose will vary depending on factors such as the age, bodyweight, health condition, sex, symptoms of a patient, the route ofadministration, the frequency of administration, the dosage form of theagent, etc. Detailed procedures for administration may be determined bythose skilled in the art. For example, for adults, the anti-tumor agentof the present invention may be administered in tablet form at a dose of0.1 μg to 10 g, preferably 1 μg to 1 g, more preferably 10 μg to 100 mg,once to five times a day.

Further, if the anti-tumor agent of the present invention is used as anagent for gene therapy, the anti-tumor agent of the present inventionmay be administered directly by injection, or alternatively, a vectorcarrying the nucleic acid may be administered. Examples of such a vectorinclude adenovirus vector, adeno-associated virus vector, herpes virusvector, vaccinia virus vector, retrovirus vector, lentivirus vector andso on. The use of these virus vectors facilitates efficientadministration.

Moreover, the anti-tumor agent of the present invention may beencapsulated within phospholipid vesicles (e.g., liposomes), and thesevesicles may be used for administration. Vesicles holding siRNA or shRNAare introduced into predetermined cells by lipofection. Then, theresulting cells are systemically administered, for example, by theintravenous or intraarterial route. They may also be topicallyadministered to the brain, etc. For example, for adults, such ananti-tumor agent may be administered at a dose of 0.1 μg/kg to 1000mg/kg per day, preferably 1 μg/kg to 100 mg/kg per day. To introducesiRNA or shRNA into a target tissue or organ, a commercially availablegene transfer kit (e.g., AdenoExpress: Clontech) may also be used.

Further, the vector expressing the polypeptide of the present inventionmay be transformed into a host (e.g., bifidobacteria, lactic acidbacteria, yeast, filamentous fungi) and the resulting transformant maybe used as an anti-tumor agent. Examples of bifidobacteria includeBifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve,etc. Examples of lactic acid bacteria include bacteria of the generaLactobacillus, Streptoccoccus, Leuconostoc, Pediococcus, etc. Such atransformant may be used directly or may be formulated into any of theabove dosage forms as appropriate before use as the anti-tumor agent ofthe present invention.

9. Angiogenesis Inhibitor and Tumor Growth Inhibitor

The polypeptide of the present invention, the vector expressing thispolypeptide, as well as shRNA and siRNA prepared in the presentinvention have an anti-angiogenic effect and an anti-tumor growtheffect. Thus, compositions comprising them can be used as angiogenesisinhibitors and as tumor growth inhibitors. Angiogenesis in the contextof the present invention is as defined above in “8. Anti-tumor agent.”

The angiogenesis inhibitor and tumor growth inhibitor of the presentinvention can be used as reagents or used for treatment of mammals, andvarious conditions including the mode of administration, the type ofadditive, the route of administration, the target to be administered,and the dose to be administered may be selected as appropriate asdescribed above in “8. Anti-tumor agent.”

10. Eliminator of Cancer Cell Characteristics

The polypeptide of the present invention and the vector expressing thispolypeptide have an elimination effect on cancer cell characteristics.Thus, compositions comprising them can be used as eliminators of cancercell characteristics. In the context of the present invention, the term“cancer cell characteristics” is intended to include the migrationcapacity, the metastatic ability, the angiogenic capacity, the propertyof growing without causing contact inhibition, the growth ability insoft agar medium (i.e., the property of growing in ananchorage-independent manner) and so on.

The eliminator of cancer cell characteristics of the present inventionmay be administered to cancer cells to thereby allow the cancer cells toacquire the ability of contact inhibition. As used herein, the term“contact inhibition” is intended to mean that normal cells stop theirgrowth at a certain level due to contact with a physical obstacle and/orcell-cell contact during their growth process. For example, when animalcells are cultured in a culture dish, contact with a physical obstacleand/or cell-cell contact will cause a change in the cytoskeleton of theanimal cells through the cell membrane to thereby change the directionof cell movement or reduce the amount of cell movement. Moreover, normalfibroblasts and normal epithelial cells will stop their growth once theyhave spread over the surface of a culture dish, unlike cancer cells.Namely, normal cells are inhibited from growing in a celldensity-dependent manner and stop their growth upon reaching a confluentstate. When they are seeded again at a low cell density, they can growagain. In contrast, cancer cells continue to grow even after the celldensity has reached a confluent state, and further continue to growuntil oxygen or nutrients are depleted. Namely, contact inhibition is anevent that is observed in normal cells, but not in cancer cells. Thus,contact inhibition is indicative of elimination of cancer cellcharacteristics. Contact inhibition also includes contact growthinhibition and contact blocking.

The elimination effect on cancer cell characteristics in the polypeptideof the present invention (e.g., a polypeptide which consists of theamino acid sequence shown in SEQ ID NO: 8, 10 or 12) and a mutantthereof may be evaluated in a known manner, for example, by introducingthe polypeptide or expression vector of the present invention intocancer cells, and determining whether the transfected cancer cells areinhibited from growing in a culture dish in a cell density-dependentmanner.

The eliminator of cancer cell characteristics of the present inventioncan be used as a reagent or used for treatment of mammals, and variousconditions including the mode of administration, the type of additive,the route of administration, the target to be administered, and the doseto be administered may be selected as appropriate as described above in“8. Anti-tumor agent.”

11. Method for Angiogenesis Inhibition and Method for Tumor GrowthInhibition

The polypeptide of the present invention, the vector expressing thispolypeptide, as well as shRNA and siRNA prepared in the presentinvention can be administered to mammals or mammalian cells to inhibitGEF-1 expression, thereby inhibiting angiogenesis and tumor growth.Thus, the present invention provides a method for angiogenesisinhibition and a method for tumor growth inhibition.

Various conditions required for the polypeptide of the present inventionand so on used in the methods of the present invention, including themode of administration, the type of additive, the route ofadministration, the target to be administered, and the dose to beadministered may be selected as appropriate as described above in “8.Anti-tumor agent.”

EXAMPLES

The present invention will be further described in more detail by way ofthe following illustrative examples, which are not intended to limit thescope of the invention.

Example 1

Preparation of GEF-1 Mutant Cells and Study of their Metastatic Ability

1. Cell Lines

Mouse melanoma cell line B16 and Lewis lung carcinoma (LLC) cells usedin this example were obtained from the Cell Resource Center forBiomedical Research, the Institute of Development, Aging and Cancer,Tohoku University, Japan, while human colorectal cancer-derived cellline COLO205, human lung cancer cell line A549, human gastriccancer-derived cell line KATOIII and human uterine cervical cancer cellline HeLa cells were obtained from the BioResource Center, RIKEN, Japan(RIKEN BRC).

The human pancreatic cancer-derived cells used were PT45 cells (Egawa N.et al., Virchows Arch. 1996 429:59-68), while the mouse vascularendothelial cells used were KOP2.16 cells (Shimamura M. et al., Int J.Cancer. 2004 111:111-6).

Canine renal epithelium-derived cell line MDCK cells and mouselymphoma-derived EL4 cells were obtained from the National Institute ofBiomedical Innovation, Japan.

The respective cells were cultured in RPMI medium (Invitrogen)containing penicillin, streptomycin and 10% FBS (HyClone) underconditions of 37° C. and 5% CO₂.

2. Preparation of GEF-1 Mutant Cells (1) Preparation of ExpressionVectors for GEF-1 and Deficient Mutants Thereof

Using primers designed for GEF-1 or each deficient mutant, i.e., a sensestrand primer containing a restriction enzyme EcoRI site and the FLAGepitope sequence as well as an antisense strand primer containing arestriction enzyme NotI site, GEF-1 was used as a template in PCR toprepare each mutant cDNA. GEF-1 of rat origin was used for this purpose.

PCR was performed for 20 cycles under conditions of denaturation at 94°C. for 30 seconds, annealing at 60° C. for 30 seconds, and elongation at72° C. for 90 seconds to 300 seconds. As primers, the following senseprimer and antisense primer were used.

Z sense primer: (SEQ ID NO: 13)5′-TTGAATTCATGGACTACAAGGACGACGATGACAAGATGGGGCGAGGC AGCGGCACC-3′P sense primer: (SEQ ID NO: 14)5′-TTGAATTCATGGACTACAAGGACGACGATGACAAGCCCCCAGAGTAC CTGACCAGC-3′C sense primer: (SEQ ID NO: 15)5′-TTGAATTCATGGACTACAAGGACGACGATGACAAGTTTAGTGAGCAG TACCAGAAC-3′Q sense primer: (SEQ ID NO: 16)5′-TTGAATTCATGGACTACAAGGACGACGATGACAAGCCCTTGCCTTAT GCCCAGCTC-3′Z-antisense primer: (SEQ ID NO: 17)5′-ATAGTTTATGCGGCCGCTAAGTGGTAGAGGCAGCTTT-3′ P-antisense primer:(SEQ ID NO: 18) 5′-ATAGTTTATGCGGCCGCTCAGGAAGTTATGGGCTGAGA-3′C-antisense primer: (SEQ ID NO: 19)5′-ATAGTTTATGCGGCCGCTAGGCACGCATCTGGACAGTCT-3′ Q-antisense primer:(SEQ ID NO: 20) 5′-ATAGTTTATGCGGCCGCTCAGTCGAAGGAGATGAGCTGGGT-3′

After the sequence of each amplification product was confirmed, eachfragment cleaved with EcoRI and NotI was introduced into the EcoRI-NotIsite of pcDNA3 animal cell expression vector (Invitrogen) to prepare anexpression vector for each of GEF-1 and Q region-deficient mutants.

(2) Preparation of GEF-1-Expressing Cells and Q Region-DeficientMutant-Expressing Cells

The following cells were prepared in a known manner (Patki, V., et al.,Nature, 394, 433-434, 1998). More specifically, the expression vectorsprepared above for GEF-1 (ZPCQ) and Q region-deficient mutants (ZPC, PCand C regions) were each introduced together with an expression vectorfor the blasticidin selection gene (pSV2bsr vector) (Funakoshi Co.,Ltd., Japan) into various cells, followed by selection with blasticidin(Funakoshi Co., Ltd., Japan) (2 mg/L) to obtain stably expressing cells.The expressing cells were each cloned by limiting dilution techniques.

a) GEF-1 (ZPCQ region)-expressing MDCK cells (MDCK/GEF-1 cells)b) GEF-1 (ZPCQ region)-expressing B16 cells (B16/GEF-1 cells)c) Q region-deficient mutant (ZPC, PC or C region)-expressing MDCK cells(MDCK/ZPC cells, MDCK/PC cells, or MDCK/C cells)d) Q region-deficient mutant (ZPC, PC or C region)-expressing B16 cells(B16/ZPC cells, B16/PC cells, or B16/C cells)e) C region-expressing LLC cells (LLC/C cells)f) C region-expressing KOP2.16 cells (KOP/C cells)g) C region-expressing PT45 cells (PT45/C cells)h) C region-expressing COLO205 cells (COLO205/C cells)i) C region-expressing EL4 cells (EL4/C cells)

For use as controls, an empty vector and pSV2bsr vector were introducedtogether to prepare control MDCK cells (MDCK/bsr cells) and control B16cells (B16/bsr cells).

(3) Preparation of B16 Cells Expressing shRNA Against the GEF-1 Genei) Preparation of shRNA Expression Vector

Expression vectors for three shRNAs (shRNA-74, shRNA-157, shRNA-207)were prepared in the following manner. It should be noted that “74”,“157” and “207” represent the positions of the 5′-terminal bases of therespective shRNAs in the open reading frame (ORF). For example, in thecase of shRNA-74, the third guanine (G) from the 5′-terminal end of theHgs74 template corresponds to the 74th base in the ORF of the GEF-1gene. The GEF-1 (Hrs/Hgs) gene used in the preparation of shRNAexpression vectors was of mouse origin.

pSuperior.neo.gfp vector (Oligoengine) was cleaved with restrictionenzymes BglII and XhoI, followed by BAP treatment.

On the other hand, three fragments to be introduced intopSuperior.neo.gfp vector were obtained by PCR. PCR was performed in areaction solution containing a Hgs template (200 pmol), a Hgs senseprimer (400 pmol), a Hgs antisense primer (400 pmol), a specificallyprepared 1× buffer, KOD DNA polymerase (2.5 U, TOYOBO), 0.2 mM dNTP and1 mM MgCl₂ under the following temperature conditions: [95° C. for 30seconds, 55° C. for 30 seconds and 68° C. for 30 seconds]×24 cycles+[95°C. for 30 seconds, 55° C. for 30 seconds and 68° C. for 7 minutes]×1cycle. The respective Hgs templates, Hgs sense primers and Hgs antisenseprimers used in PCR are shown below.

Hgs74 sense primer: (SEQ ID NO: 21) ATATAGATCTCCGGGAGTCCATTCTACAGATHgs74 antisense primer: (SEQ ID NO: 22) GGTACCTCGAGTAAAAAGGGAGTCCAHgs74 template: (SEQ ID NO: 23)CCGGGAGTCCATTCTACAGATTTCAAGAGAATCTGTAGAATGGACTCCCT TTTTAHgs157 sense primer: (SEQ ID NO: 24) ATATAGATCTCCGTTAATGATAAGAACCHgS157 antisense primer: (SEQ ID NO: 25) GGTACCTCGAGCTTAAAAAGTTAATGATHgs157 template: (SEQ ID NO: 26)CCGTTAATGATAAGAACCCACTTCAAGAGAGTGGGTTCTTATCATTAACT TTTTAHgs207 sense primer: (SEQ ID NO: 27) ATATAGATCTCCGTCTGTGGTAAAGAACTHgs207 antisense primer: (SEQ ID NO: 28) GGTACCTCGAGCTTAAAAAGTCTGTGGTHgs207 template: (SEQ ID NO: 29)CCGTCTGTGGTAAAGAACTGTTTCAAGAGAACAGTTCTTTACCACAGACT TTTTA

The above three fragments obtained by PCR were purified and then cleavedwith restriction enzymes BglII and XhoI, and each introduced into theBAP-treated pSuperior.neo.gfp vector.

ii) Preparation of shRNA-Expressing B16 Cells

The respective shRNA expression vectors prepared in i) above were eachintroduced into B16 cells by gene transfer and subjected to drugselection using G418 drug (1 mg/ml). B16 cells expressing the respectiveshRNAs (B16/shRNA-74 cells, B16/shRNA-157 cells and B16/shRNA-207 cells)were finally obtained by cloning. These cells were confirmed for theirGEF-1 expression levels by Western blotting and also examined for theirability of cell migration using a Boyden chamber.

As a result, the three types of cells all showed 80% or more inhibitionof both Hgs expression and cell migration ability. The B16/shRNA-157cells showed 95% inhibition of both Hgs expression and cell migrationability, and hence showed the highest inhibition among the three typesof cells. Then, B16/shRNA-157 cells were used as B16/shRNA cells in thesubsequent experiments.

Among the cells prepared in (2) and (3) above, B16/bsr cells, B16/GEF-1cells, B16/C cells, B16/shRNA cells and their parent B 16 cells aremorphologically shown in FIG. 2A.

As shown in FIG. 2A, the B16/GEF-1 cells showed a more fibroblast-likemorphology with less cell-cell contact than their parent B16 cells orthe B16/bsr cells. In contrast, the B16/C cells and the B16/shRNA cellsshowed a morphology with more cell-cell contact.

This result indicated that expression of GEF-1 enhanced the migrationcapacity of B16 cells, whereas expression of the C region and shRNA ofGEF-1 inhibited the migration capacity of B16 cells.

Further, the B16/GEF-1 cells, the B16/C cells, the B16/shRNA cells andthe B16 cells were examined for their metastatic ability. Morespecifically, the B16/GEF-1 cells, the B16/C cells, the B16/shRNA cellsand the B16 cells (1×10⁶/0.2 mL PBS) were each administered to C57BL6mice via the tail vein, and metastasis in their lungs were observed at21 days after administration. In addition, HE staining was alsoconducted. Further, the number of days from cell administration untilthe death of each animal was counted to determine the number of days toreach 50% mortality.

As a result, in the mice at 21 days after administration, the B16/GEF-1cells enhanced their metastasis to the lung, whereas the B16/shRNA andB16/C cells were inhibited from metastasizing to the lung. Inparticular, the B 16/C cells were significantly inhibited frommetastasizing to the lung (FIG. 2B).

Moreover, the 50% longevity (survival days) was 42 days in the B16 cellsand the B16/bsr cells, whereas the 50% longevity was greatly reduced to25 days in the B16/GEF-1 cells. In contrast, the 50% longevity was 87days in the B16/C cells, which was increased twice or more than in theB16 cells and the B16/bsr cells. Likewise, the B16/shRNA cells alsoshowed a great increase in 50% longevity as compared to the B16 cellsand the B16/bsr cells (FIG. 2C).

These results indicated that expression of GEF-1 enhanced metastasis ofcancer cells, whereas expression of the C region and shRNA of GEF-1significantly inhibited metastasis of cancer cells.

Example 2

Study of Polypeptides Covering the C Region of GEF-1 for their Effect onAngiogenesis

1. In Vitro Tube Formation Test

KOP2.16 cells were used as vascular endothelial cells. KOP2.16 cellswere modified to stably express the C region of GEF-1 (KOP/C cells) andmeasured for their tube formation ability.

More specifically, the above KOP2.16 cells and KOP/C cells were eachadjusted to fixed cell counts and seeded on Matrigel in a microplate.After culture for 18 to 48 hours, the cells were photographed under aninverted microscope. Based on the resulting images, the state of tubeformation was scored using measurement software to determine the tubeformation ability.

As a result, the mouse vascular endothelial cells (KOP2.16) showed atube morphology where the cells were assembled, whereas the tubeformation ability disappeared in the KOP/C cells (FIG. 3A).

Moreover, MDCK cells, which are renal epithelium-derived cells, but notvascular endothelium-derived cells, were also subjected to the same tubeformation test as in the case of the KOP cells, because MDCK cellsslightly have the tube formation ability.

As a result, MDCK/GEF-1 cells significantly enhanced their tubeformation ability. In contrast, the tube formation ability completelydisappeared in MDCK/C cells (FIG. 3B).

These results indicated that polypeptides comprising the C region ofGEF-1 inhibited tube formation during angiogenesis.

2. In Vivo Angiogenesis Induction Test

Using an in vivo dorsal air sac (DAS) assay, B16 cells were examined fortheir ability to induce angiogenesis. The DAS assay is an assay used forstudy of pathological angiogenesis including tumor-induced angiogenesis.

Chambers holding the respective cells (B16 cells, B16/GEF-1 cells, B16/Ccells, B16/shRNA cells) were transplanted under the dorsal skin of mice.After 7 days, each chamber was removed and the dorsal skin of each mousewas then photographed under a stereoscopic microscope. Based on theresulting images, the number and area of neovascular vessels induced bythe tumor cells were scored using measurement software to determine thetube formation ability.

As a result, the B16/GEF-1 cells enhanced their ability to induceangiogenesis as compared to the B16 cells. In contrast, the B16/C cellsand the B16/shRNA cells showed a reduction in their ability to induceangiogenesis (FIG. 4).

These results indicated that expression of GEF-1 induced, both in vitroand in vivo, cancer cell-induced angiogenesis (pathologicalangiogenesis) and vascular endothelial cell angiogenesis.

Moreover, the polypeptides (polypeptides comprising the C region ofGEF-1) and inhibitory substances (shRNAs against a polynucleotideencoding GEF-1) in the present invention were found to inhibit cancercell-induced angiogenesis and vascular endothelial cell angiogenesis.

Example 3

1. Study of Polypeptides Covering the C Region of GEF-1 for their Effecton Tumor Growth

(1) In Vitro Growth of B 16/C Cells and EL4/C Cells

The relationship between GEF-1 expression and tumor growth was studiedusing GEF-1 mutant cell lines of B16 cells and EL4 cells.

The respective cells (B16 cells, B16/GEF-1 cells, B16/C cells, B16/shRNAcells, EL4 cells, EL4/C cells) were washed with PBS and then treatedwith TrypLE Express® (Invitrogen) to separate the cells, which were thensuspended again in Opti-MEM I® Reduced-Serum Medium (Opti-MEM I medium,Invitrogen). Cell counts in these suspensions were measured using aCountess®.

As a result, the B16/GEF-1 cells and the B16/shRNA cells showed the samegrowth pattern as that of their parent B16 cells. These cells all diedwhen the cell density reached about 3×10⁶/cm².

In contrast, the B16/C cells reached a confluent state and stopped cellgrowth when the cell density reached 5×10⁵/cm² (FIG. 5). When seededagain at a low cell density, these B16/C cells were found to show thesame growth ability as before. Likewise, the EL4 cells all died when thecell density reached about 9×10⁵/cm². In contrast, the EL4/C cellsreached a confluent state and stopped cell growth when the cell densityreached 6×10⁵/cm². The EL4/C cells were also found to stop cell growthin a cell density-dependent manner (FIG. 25).

These results indicated that the B16/C and EL4/C cells each expressing apolypeptide covering the C region of GEF-1 had the ability to inhibitcontact growth in a cell density-dependent manner, as observed in normalcells, and their cancer cell characteristics disappeared.

(2) Cell Growth and Cell Cycle of B 16/C Cells

To study polypeptides covering the C region of GEF-1 for their effect oncell cycle, the B 16/C cells were analyzed for their cell cycle by FACSassay. More specifically, the cells prepared with a CycleTest® Plus kit(Becton Dickinson (hereinafter referred to as BD)) were analyzed with aFACS calibur (BD).

As a result, the B16/C cells were found to show G1 phase:S phase:G2/Mphase=41:51:8 during growth phase at a low cell density, 72:21:7 uponreaching a confluent state, and 90:2:8 at 4 weeks after reaching aconfluent state (FIG. 6).

This result indicated that the B16/C cells were arrested in the G1 phasein a cell density-dependent manner and hence entered G1 arrest, so thattheir growth ability was significantly reduced and their cancer cellcharacteristics disappeared.

2. DNA Microarray Analysis and Profiling Analysis

To clarify the cell characteristics of the B16/GEF-1 cells, theB16/shRNA cells, the B16/C cells and the B16 cells, mRNAs were extractedfrom the respective cells and subjected to DNA microarray (AgilentTechnologies) analysis. Further, analysis software KeyMolnet (IMMD Inc.,Japan) was used to profile the resulting data.

The results obtained are shown in the table below.

Cell Rank Pathway based on Molecule Score B16/GEF-1 1 SMAD-mediatedexpression regulation 78.3 2 RB/E2F-mediated expression regulation 64.7B16/shRNA 1 RB/E2F-mediated expression regulation 235.4 2 SMAD-mediatedexpression regulation 84.8 B16/C 1 RB/E2F-mediated expression regulation197.1 2 SMAD-mediated expression regulation 97.2

This result indicated that the B16/GEF-1 cells, the B16/shRNA cells andthe B16/C cells showed great changes in protein expression downstream ofthe SMAD pathway and the Rb/E2F pathway, as compared to their parent B16cells.

This result also indicated that changes in intracellular GEF-1expression levels affected protein expression downstream of theTGF-β-SMAD signaling pathway and the Rb/E2F signaling pathway throughthese pathways. The TGF-β-SMAD signaling pathway is greatly involved incell growth, differentiation and apoptosis. Rb is among the most majortumor suppressor gene products, as in the case of p53. Rb also has thefunction of controlling the cell cycle through binding to E2F.

Thus, the above results suggested that regulation of GEF-1 expressionlevels would allow regulation of the TGF-β-SMAD signaling pathway andthe Rb/E2F signaling pathway, and further would allow control orinhibition of tumor cell growth.

Further, in experiments using MDCK cells, MDCK/GEF-1 cells showedreduced levels of SMAD7 and Rb expression, whereas MDCK/C cells showedincreased levels of Rb expression and reduced levels of Cyclin A, D, E,p53, c-Ski, CTGF (connective tissue growth factor), HDAC4 and ATMexpression. Likewise, MDCK/shRNA cells showed increased levels of c-Mycand c-Fos expression and reduced levels of Cyclin A, B, D, E and AuroraB expression.

3. Activity Analysis of Transcriptional Regulatory Factors

The results of the above DNA array analysis suggested that changes inGEF-1 expression levels would affect the activity of SMAD and Rbtranscription factors. Then, various transcription factors were examinedfor their activity and other properties in the MDCK/GEF-1 cells and theMDCK/C cells.

(1) Effects of HGF and TGF-β on SMAD Transcriptional Activity inMDCK/GEF-1 Cells and MDCK/C Cells

For measurement of SMAD transcriptional activity, pSmad RE-TK hRluc(F)and phRL-TK (both available from RIKEN BRC)) were used. pSmad RE-TKhRluc(F) is a vector having each response element (cis-acting DNAsequence) upstream of a TATA-like promoter and the luciferase gene as areporter gene downstream of the TATA-like promoter. A schematic view ofthis vector is shown in FIG. 7.

The MDCK/GEF-1 cells and the MDCK/C cells were each seeded in 48-wellplates at 5.0×10⁴ cells per well. After 24 hours, the cells were washedwith Opti-MEM medium, and an Opti-MEM medium mixture (100 μl) containing0.5 μg of pSmad-RE-TK-hRluc(F), 0.05 μg of pGL4.13 (Promega) and 0.5 μlof Lipofectamin 2000 (Invitrogen) was added to the cells to effect genetransfer. After culture in a CO₂ incubator for 4 hours, 0.4 ml ofOpti-MEM medium was added. At 24 hours after gene transfer, the mediumwas replaced with Opti-MEM medium containing HGF (40 ng/ml, PeproTechEC) or TGF-β (5 ng/ml, PeproTech EC). At 48 hours after gene transfer,the cells were washed with PBS and measured for luciferase activity witha system for dual-luciferase quantification.

As a result, the SMAD transcriptional activity in the MDCK/GEF-1 cellswas enhanced about 570 times, as compared to MDCK cells.

The SMAD transcriptional activity in the MDCK cells was enhanced abouttwice upon HGF stimulation, whereas no further enhancement was observedin the MDCK/GEF-1 cells. Moreover, upon TGF-β stimulation, the SMADtranscriptional activity was enhanced about 11 times in the MDCK cellsand about 6.4 times in the MDCK/GEF-1 cells.

In contrast, the SMAD transcriptional activity in the MDCK/C cells wasinhibited as compared to the SMAD transcriptional activity in the MDCKcells. Further, even upon HGF stimulation or TGF-β stimulation, theMDCK/C cells showed no enhancement of the SMAD transcriptional activity(FIG. 8).

This result indicated that upon expression of polypeptides covering theC region of GEF-1, the SMAD transcriptional activity in MDCK cells wasinhibited and was also no longer affected by HGF stimulation or TGF-βstimulation.

(2) Transcription Factor Activity in MDCK Mutant Cells

In the above study, the SMAD transcriptional activity was enhanced inthe MDCK/GEF-1 cells and inhibited in the MDCK/C cells, as compared tothe MDCK cells. Then, further study was conducted to measure theactivity of transcription factors related to various signaling pathwaysin the MDCK/GEF-1 cells and the MDCK/C cells.

For measurement of the transcriptional activity of various transcriptionfactors including Rb and p53, a BD Pathway Profiling Luciferase System(BD) and a BD Pathway Profiling Luciferase System 4 (BD) were used.

As a result, the respective cells were controlled to enhance or inhibitthe expression of proteins downstream of SMAD. Particularly in theMDCK/C cells, the p53 transcriptional activity was enhanced while the Rbtranscriptional activity was inhibited (FIG. 9). A reduction in the Rbtranscriptional activity is indicative of an increase in E2F/Rb complexlevels.

(3) Transcription Factor Activity in B16 Mutant Cells

The same procedure as used above for MDCK cells was repeated to measurethe activity of transcription factors related to various signalingpathways in the B16/GEF-1 cells and the B16/C cells.

As a result, the SMAD transcriptional activity was enhanced in theB16/GEF-1 cells, whereas the SMAD transcriptional activity and the Rbtranscriptional activity were inhibited and the p53 transcriptionalactivity was enhanced in the B16/C cells (FIG. 10). A reduction in theRb transcriptional activity is indicative of an increase in E2F/Rbcomplex levels.

As shown above, the MDCK/C cells and the B16/C cells both showed reducedRb transcriptional activity and enhanced p53 transcriptional activity. Areduction in the Rb transcriptional activity is indicative of anincrease in Rb/E2F complex formation, which promotes cell cycle arrestin the G1 phase. In addition, enhanced p53 transcriptional activityinduces p21 expression and, in turn, p21 inhibits Rb phosphorylation,whereby the cell cycle is arrested in the G1 phase.

Thus, the above results indicated that upon expression of polypeptidescovering the C region of GEF-1, E2F/Rb complex formation and p53expression were enhanced, which in turn would arrest the cell cycle inthe G1 phase and hence inhibit cell growth.

(4) PAI-1 Promoter Activity in B16 Mutant Cells

μPlasminogen activator inhibitor-1 (PAI-1) is known to be induced toexpress itself by the SMAD transcription factor. Based on thisknowledge, TGF-β-SMAD signaling levels in the MDCK/GEF-1 cells and theMDCK/C cells were determined by measuring the promoter activity ofPAI-1. The promoter activity of PAI-1 was measured in the same manner asshown in (1) above by measuring luciferase activity with a system fordual-luciferase quantification. A schematic view of a PAI-1promoter-carrying vector is shown in FIG. 7.

As a result, the PAI-1 promoter activity was increased about four timesin the B16/GEF-1 cells and reduced to 3/4 in the B16/C cells, ascompared to the B16 cells (FIG. 11).

This result indicated that TGF-β-SMAD signaling was enhanced about fourtimes in the B16/GEF-1 cells and inhibited to 3/4 in the B 16/C cells,as compared to the B16 cells.

4. Measurement of TGF-β Expression Levels

TGF-β-SMAD signals are transmitted to intracellular SMAD moleculesthrough binding of extracellular TGF-β to its receptors on the cellsurface. Then, the SMAD molecules migrate into the nuclei and bind toDNA, thereby enhancing the transcriptional activity of proteinscorresponding to TGF-β-SMAD signaling. Moreover, TGF-β molecules per seare also protein molecules whose expression is induced by TGF-β-SMADsignaling, so that enhanced TGF-β-SMAD signaling also enhances TGF-βprotein synthesis and extracellular release. Thus, TGF-β levels in acell culture are proportional to TGF-β-SMAD signaling levels in thecells.

Then, TGF-β expression levels in a cell culture were measured andanalyzed with a Quantikine® TGF-β ELISA assay kit (R&D Systems, Inc.).

As a result, protein levels of TGF-β were increased in cultures of theB16 cells and the B16/GEF-1 cells, whereas they were reduced in culturesof the B16/C cells and the B16/shRNA cells (FIG. 12).

Thus, TGF-β-SMAD signaling would have been enhanced in the B16 cells andthe B16/GEF-1 cells and would have been inhibited in the B16/C cells.TGF-β levels in the B16/GEF-1 cell culture were about twice higher thanthose of the B16 cell culture, thus suggesting that TGF-β-SMAD signalinglevels in the B16/GEF-1 cells would be enhanced about twice, as comparedto the B16 cells.

5. Measurement of In Vitro Cell Growth Ability in Soft Agar

Anchorage-independent growth ability is one of the cancer cellcharacteristics. Normal (non-cancer) cells can grow only in ananchorage-dependent manner and hence cannot grow in soft agar medium. Incontrast, cancer cells can grow in soft agar medium to form colonies.Then, the respective cells were measured for their growth ability insoft agar medium.

A cell culture solution (1.5 ml), in which agarose (low gellingtemperature agarose) was dissolved at a concentration of 0.5% (w/v), wasadded to a 35 mm dish, cooled and gelled. Onto the resulting gel, a0.33% agarose/cell culture medium containing 5.0×10³ cells was added ina volume of 1.5 ml. After gelling, the cells were cultured in a CO₂incubator for 2 weeks. A 0.005% (w/v) aqueous solution of crystal violet(0.5 ml) was added onto the gel and incubated overnight to stain thecells. The 35 mm dish was scanned with a scanner, and colony counts weremeasured from the resulting image using image analysis software.

As a result, the growth ability of the B16/GEF-1 cells in soft agarmedium was enhanced about twice, as compared to the B 16 cells, whereasthe growth ability of the B16/C cells in soft agar medium was reduced toless than 5% of that of the B16 cells (FIG. 13A).

In the same manner, a stably GEF-1/C protein-expressing cell line ofmouse lung cancer-derived LLC cells (LLC/C cells), a stably GEF-1/Cprotein-expressing cell line of human pancreatic cancer-derived cellline PT45 cells (PT45/C cells), and a stably GEF-1/C protein-expressingcell line of human colorectal cancer-derived cell line COLO205 cells(COLO205/C cells) were measured for their growth ability in soft agarmedium.

As a result, the growth ability in soft agar medium was reduced to lessthan 20% in the LLC/C cells, as compared to their parent LLC cells (FIG.13B). Likewise, the growth ability in soft agar medium was reduced toless than 5% in the PT45/C cells, as compared to their parent PT45 cells(FIG. 26), while the growth ability in soft agar medium was reduced toless than 1% in the COLO205/C cells, as compared to their parent COLO205cells (FIG. 27).

Next, MDCK cells were measured for their growth ability in soft agarmedium. MDCK cells, which are derived from canine renal epithelialcells, are not cancerous and hence retain a normal cell-like morphology.Thus, MDCK cells cannot grow in soft agar medium. However, a stablyGEF-1 protein-expressing cell line of MDCK cells (MDCK/GEF-1 cells)acquired the growth ability in soft agar medium (FIG. 13C).

These results indicated that when GEF-1 was expressed in B16 cells andLLC cells, both of which are cancer cells, their growth ability in softagar medium was enhanced, whereas GEF-1 expression in MDCK cells, whichare not cancer cells, allowed the MDCK cells to acquire the growthability in soft agar medium, which is not inherent to the MDCK cells. Onthe other hand, when polypeptides covering the C region of GEF-1 wereexpressed in B16 cells, LLC cells, PT45/C cells and COLO205/C cells,there was a significant reduction in their tumor growth ability in softagar medium.

Namely, it was indicated that when polypeptides covering the C region ofGEF-1 were expressed in B16 cells, LLC cells, PT45/C cells and COLO205/Ccells, these cells lost their anchorage-independent growth ability,which is among the cancer cell characteristics.

6. In Vivo Tumorigenicity Test

The respective cells (B16 cells, B16/GEF-1 cells, B16/C cells, B16/shRNAcells) were each transplanted subcutaneously into C57BL/6 mice, and thetumor volume was measured at intervals of a week.

C57BL/6 mice were shaved and inoculated subcutaneously with therespective cells (2.0×10⁶ cells/0.2 ml PBS). The long and short axes oftumor were measured at intervals of a week to calculate the tumor volume(=long axis×(short axis)²×π/6).

As a result, the B16/GEF-1 cells showed about 2-fold enhancement oftheir subcutaneous tumorigenicity in C57BL/6 mice, as compared to theB16 cells. In contrast, the B16/C cells and the B16/shRNA cells bothshowed a reduction in their tumorigenicity (FIG. 14A).

This result indicated that the B16/GEF-1 cells had higher in vivotumorigenicity than the B16 cells. In contrast, the B16/C cells and theB16/shRNA cells were both found to have suppressed tumorigenicity.

Further, to measure MDCK cells and MDCK/GEF-1 cells for their in vivotumorigenicity, these cells (1.0×10⁶ cells/0.2 ml PBS) were transplantedsubcutaneously into nude mice. At 4 weeks after transplantation, themice were euthanized with carbon dioxide gas, and tumor was then excisedfrom each mouse and measured for its wet weight.

As a result, the MDCK cells formed no tumor under the skin of nude micebecause they were not cancerous and retained a normal cell-likemorphology. In contrast, the MDCK/GEF-1 cells formed tumor under theskin of nude mice (FIG. 14B).

In the above in vivo tumorigenicity test, the B16/GEF-1 cells and theMDCK/GEF-1 cells showed enhancement of their tumor growth ability. Onthe other hand, the B16/C cells and the B16/shRNA cells showed areduction in their tumor growth ability. These results indicated thatincreased intracellular levels of GEF-1 protein enhanced tumor growthability, whereas suppressed levels of GEF-1 protein inhibited tumorgrowth ability. Thus, it was indicated that when controlling theexpression and functions of GEF-1, the tumor growth ability of cancercells can be controlled.

Moreover, upon expression of polypeptides covering the C region ofGEF-1, B16 cells and LLC cells showed a reduction in their tumor growthability, indicating that the polypeptides of the present inventioninhibited tumor growth.

Example 4 Preparation of GEF-1-Related Small Molecules

The results of Example 3 described above indicated that tumor growth canbe inhibited when the expression levels of GEF-1 are suppressed or whenthe GEF-1/C protein is expressed to inhibit GEF-1 functions.

The above B16/shRNA cells are derived from B16 cells by introducing aninterference-inducing vector to suppress the expression levels of GEF-1by RNA interference effects. Likewise, the B16/C cells are derived fromB16 cells by introducing a GEF-1/C protein expression vector to inducethe expression of GEF-1/C protein. The RNA interference-inducing vectorand the GEF-1/C protein expression vector are both macromolecules andhence are difficult to introduce into cancer cells in the human body andare not easy to use in clinical practice.

However, small molecule siRNAs capable of inducing RNA interference andGEF-1/C region constituent oligopeptides are easier to introduce ordeliver (DDS) into cancer cells in the human body and can also beexpected to be used in clinical practice.

Then, further study was conducted to investigate a method for tumorgrowth inhibition by GEF-1-related small molecules targeting GEF-1,i.e., siRNAs and GEF-1/C region constituent oligopeptides.

1. siRNAs

For use as siRNAs targeting GEF-1 (GEF-1-siRNAs), siRNAs (stealth RNAi)against mouse Hgs (NM_(—)008244) were synthesized by entrusting theirsynthesis to Invitrogen. The nucleotide sequences of six siRNAs (siRNA#1to #6) and siRNA#3-scramble are shown below. It should be noted thatsiRNA#3-scramble is intended to mean scramble siRNA of siRNA#3, i.e., aduplex RNA whose nucleotide sequence is different from that of siRNA#3,but whose ratio of constituent nucleotides is equal to that of siRNA#3,and whose homologous sequence is not found in cells or animals to beused. siRNA#3-scramble was used as a control for siRNA#3.

siRNA #1 NM_008244_stealth_1064 Sense strand: (SEQ ID NO: 30)5′-ACCUCCACGUCUCUGUCGAUCGAUA Antisense strand: (SEQ ID NO: 31)5′-UAUCGAUCGACAGAGACGUGGAGGU siRNA #2 NM_008244_stealth_677Sense strand: (SEQ ID NO: 32) 5′-GGACGAUACUCGUCGACUUGUUCUUAntisense strand: (SEQ ID NO: 33) 5′-AAGAACAAGUCGACGAGUAUCGUCC siRNA #3NM_008244_stealth_99 Sense strand: (SEQ ID NO: 34)5′-UAGAAUGGACUCCCAGUCUGACUCC Antisense strand: (SEQ ID NO: 35)5′-GGAGUCAGACUGGGAGUCCAUUCUA siRNA #3 scrambleNM_008244_stealth_scramble_99 Sense strand: (SEQ ID NO: 36)5′-UAGCAAUAGGCACUCCGACUGUUCC Antisense strand: (SEQ ID NO: 37)5′-GGAACAGUCGGAGUGCCUAUUGCUA siRNA #4 NM_008244_stealth_74 Sense strand:(SEQ ID NO: 38) 5′-AACAGAAGCUGGCUGGUGGCUUUGU Antisense strand:(SEQ ID NO: 39) 5′-ACAAAGCCACCAGCCAGCUUCUGUU siRNA #5NM_008244_stealth_255 Sense strand: (SEQ ID NO: 40)5′-AUCAUGGACUGUCUGGCCACAGUUC Antisense strand: (SEQ ID NO: 41)5′-GAACUGUGGCCAGACAGUCCAUGAU siRNA #6 NM_008244_stealth_1026Sense strand: (SEQ ID NO: 42) 5′-UUUCUUCUCCCAGUAGUUCCGGUUGAntisense strand: (SEQ ID NO: 43) 5′-CAACCGGAACUACUGGGAGAAGAAA

2. GEF-1/C Constituent Oligopeptides

The GEF-1/C constituent oligopeptides used in this example weresynthesized by entrusting their synthesis to Hayashi-Kasei Co., Ltd.,Japan.

A schematic view of the GEF-1/C constituent oligopeptides is shown inFIG. 17. The amino acid sequences of the synthesized oligopeptides arealso shown below. Among the synthesized oligopeptides shown below,r9-OP10-11 is a peptide composed of OP10-11 linked to a cell membranepermeable peptide (rrrrrrrrrGPG (where r represents D-arginine) (SEQ IDNO: 66)). Although the GEF-1/C constituent oligopeptides shown below areof mouse origin, they differ from oligopeptides of human origin only inT (G in human) at position 16 from the N-terminal end of OP20-2 (atposition 6 from the N-terminal end of OP-20-3 or OP10-3), and the otherresidues are common to the mouse and human oligopeptides.

(SEQ ID NO: 44) OP20-1: MKSNHMRGRSITNDSAVLSL (SEQ ID NO: 45) OP20-2:ITNDSAVLSLFQSINTMHPQ (SEQ ID NO: 46) OP20-3: FQSINTMHPQLLELLNQLDE(SEQ ID NO: 47) OP20-4: LLELLNQLDERRLYYEGLQD (SEQ ID NO: 48) OP20-5:RRLYYEGLQDKLAQIRDARG (SEQ ID NO: 49) OP20-6: KLAQIRDARGALSALREEHR(SEQ ID NO: 50) OP20-7: ALSALREEHREKLRRAAEEA (SEQ ID NO: 51) OP20-8:EKLRRAAEEAERQRQIQLAQ (SEQ ID NO: 52) OP20-9: ERQRQIQLAQKLEIMRQKKQ(SEQ ID NO: 53) OP20-10: KLEIMRQKKQEYLEVQRQLA (SEQ ID NO: 54) OP20-11:EYLEVQRQLAIQRLQEQEKE (SEQ ID NO: 55) OP20-12: IQRLQEQEKERQMRLEQQKQ(SEQ ID NO: 56) OP20-13: RQMRLEQQKQTVQMRAQMPA (SEQ ID NO: 57) OP10-1:MGRGSGTFER (SEQ ID NO: 58) OP10-3: FQSINTMHPQ (SEQ ID NO: 59) OP10-6:KLAQIRDARG (SEQ ID NO: 60) OP10-7: ALSALREEHR (SEQ ID NO: 61) OP10-8:EKLRRAAEEA (SEQ ID NO: 62) OP10-9: ERQRQIQLAQ (SEQ ID NO: 63) OP10-10:KLEIMRQKKQ (SEQ ID NO: 64) OP10-11: EYLEVQRQLA (SEQ ID NO: 65) OP10-12:IQRLQEQEKE (SEQ ID NO: 67) r9-OP10-11: rrrrrrrrrGPGEYLEVQRQLA

Example 5 Inhibition of Angiogenesis and Tumor Growth by GEF-1-RelatedSmall Molecules

1. siRNA-Induced Inhibition of Angiogenesis and Tumor Growth(1) Study of siRNA-Induced Angiogenesis Inhibition

To confirm the in vitro anti-angiogenic effect of the GEF-1-siRNAs, atube formation inhibition test was conducted using vascular endothelialKOP2.16 cells.

Each siRNA (40 nmol) and lipofectamine 2000 (10 μl) were mixed and thendiluted to 1.0 ml with Opti-MEM I medium, and added to KOP2.16 cells ina 35 mm dish, which had been washed with Opti-MEM I medium. After 4hours, 20% FBS-containing Opti-MEM I medium (1.0 ml) was added to thedish. After 48 hours, siRNA-transfected cells were collected andcounted, and then suspended in Opti-MEM I medium and seeded again onMatrigel. After 18 to 48 hours, tube formation in the cells wasobserved. As negative controls, PBS (“None” in FIG. 15) and Stealth RNANegative Control Medium GC Duplex (Invitrogen) (“siRNA-MNC” in FIG. 15)were used.

As a result, among the six siRNAs, siRNA#2 and siRNA#3 showed asignificant inhibitory effect on tube formation (FIG. 15).

This result indicated that the above siRNAs showed an anti-angiogeniceffect through inhibition of GEF-1 expression.

(2) Study of siRNA-Induced Tumor Growth Inhibition

Further, siRNA#3 showing the highest inhibitory effect on tube formationin (1) above was examined for its anti-tumor growth effect.

B 16 cells were inoculated subcutaneously into nude mice. After about aweek when tumor reached about 5 mm diameter (about 100 m³), ansiRNA#3/atelocollagen mixture (0.2 ml) was injected near the tumor.Subsequently, the same siRNA#3/atelocollagen mixture was furtherinjected twice near the tumor at intervals of a week.

As a result, siRNA#3 was able to suppress the tumor volume and the tumorweight to 50% or less, as compared to the case where PBS or scramblesiRNA of siRNA#3 (siRNA#3-scramble) was administered (FIG. 16).

This result indicated that the above siRNA showed an in vivo anti-tumorgrowth effect through inhibition of GEF-1 expression.

2. Inhibition of Angiogenesis and Tumor Growth by GEF-1/C ConstituentOligopeptides (1) Effect of GEF-1/C Constituent Oligopeptides on SMADTranscriptional Activity

First, the GEF-1/C constituent oligopeptides were studied for theireffect on the SMAD transcriptional activity in TGF-β-SMAD signaling. TheTGF-β-SMAD signaling pathway is greatly involved in angiogenesis andcancer cell metastasis.

Each GEF-1/C constituent oligopeptide (2.5 nmol) and a peptidetransporter Wr-T (0.2 nmol) were mixed and then diluted to 0.1 ml withPBS, and mixed with a DNA solution for SMAD transcriptional activitytesting (0.1 ml). To the respective cells (B16 cells and COLO205 cells)in a 48-well plate, which had been washed with Opti-MEM I medium, themixture thus prepared was added in a volume of 0.2 ml per well. After 4hours, 20% FBS/Opti-MEM I medium (0.2 ml) was added to each well. After48 hours, the respective cells were lysed and the luciferase activity ineach lysate was measured.

As a result, in the B16 cells, the GEF-1/C constituent oligopeptidesOP10-7, OP10-11 and OP10-12 caused about 50% inhibition of the SMADtranscriptional activity (FIG. 18A).

Likewise, in the human colorectal cancer COLO205 cells, OP10-6 toOP10-12 caused about 35% to 70% inhibition of the SMAD transcriptionalactivity (FIG. 18B).

(2) Study of GEF-1/C Constituent Oligopeptide-Induced AngiogenesisInhibition

To confirm the inhibitory effect of the GEF-1/C constituentoligopeptides on in vitro angiogenesis, a tube formation inhibition testwas conducted using vascular endothelial KOP2.16 cells.

Each GEF-1/C constituent oligopeptide (2.5 nmol) and a peptidetransporter Wr-T (0.2 nmol) were mixed and then diluted to 0.1 ml withOpti-MEM I medium, and added to KOP2.16 cells in a 48-well plate, whichhad been washed with Opti-MEM I medium. After 2 hours, 10%FBS-containing Opti-MEM I medium (0.4 ml) was added to each well. After48 hours, GEF-1/C constituent oligopeptide-transfected cells werecollected and counted, and then suspended in Opti-MEM I medium. Thesecells were seeded on Matrigel.

As a result, OP10-10 to OP10-12 caused about 50% inhibition of tubeformation (FIG. 19).

This result indicated that the above GEF-1/C constituent oligopeptidesshowed an anti-angiogenic effect.

(3) Study of GEF-1/C Constituent Oligopeptides for their In VitroAnti-Tumor Growth Effect

The GEF-1/C constituent oligopeptides were studied for their inhibitoryeffect on the growth of various tumor cells (B16 cells, HeLa cells,COLO205 cells, KATO III cells, A549 cells and PT45 cells) in soft agarmedium.

In the same manner as shown in (2) above, the GEF-1/C constituentoligopeptides were introduced into various tumor cells and these cellswere seeded together with a 0.33% soft agar medium.

The results obtained are shown below for the respective cells.

a) Mouse melanoma B16 cells

OP10-6 to OP10-12 caused about 30% or more inhibition of the B16 cells'growth ability in soft agar medium. In particular, OP10-11 caused about60% inhibition (FIG. 20A).

b) Human uterine cervical cancer-derived HeLa cells

OP10-11 caused about 20% inhibition of the HeLa cells' growth ability insoft agar medium (FIG. 20B).

c) Human colorectal cancer-derived COLO205 cells

OP10-10 and OP10-11 caused about 50% inhibition of the COLO205 cells'growth ability in soft agar medium (FIG. 20C).

d) Human gastric cancer-derived KATO III cells

OP10-10 and OP10-11 caused about 50% to 70% inhibition of the KATO IIIcells' growth ability in soft agar medium (FIG. 20D).

e) Human lung cancer-derived A549 cells

OP10-7, OP10-11 and OP10-12 caused about 40% to 50% inhibition of theA549 cells' growth ability in soft agar medium (FIG. 21A).

f) Human pancreatic cancer-derived PT45 cells

OP10-7, OP10-11 and OP10-12 caused about 30% to 60% inhibition of thePT45 cells' growth ability in soft agar medium (FIG. 21B).

(4) Study of GEF-1/C Constituent Oligopeptides for their In VivoAnti-Tumor Growth Effect

Next, the GEF-1/C constituent oligopeptides were studied for their invivo anti-tumor growth effect.

Each GEF-1/C constituent oligopeptide (25 nmol) and a peptidetransporter Wr-T (2 nmol) were mixed and then diluted to 0.1 ml withPBS, to which each cell suspension prepared at 5.0×10⁶ cells/ml Opti-MEMI medium was then added in a volume of 0.1 ml and mixed. This cellmixture was incubated in a CO₂ incubator at 37° C. for 2 hours and theninoculated subcutaneously into nude mice. The long and short axes oftumor were measured at intervals of a week to calculate the tumor volume(=long axis×(short axis)²×π/6). After the mice were finally euthanizedwith carbon dioxide gas, tumor in each mouse was photographed.Subsequently, the tumor was excised and measured for its wet weight.

The results obtained are shown below for the respective tumor cells.Since OP10-11 showed the highest inhibitory effects on tube formationand growth ability in soft agar medium in the above in vitro tests,OP10-11 was also used in the in vivo test. It should be noted that #8 to#12 in FIGS. 23 and 24 represent OP10-8 to OP10-12, respectively.

a) Mouse melanoma B16 cells

OP10-11 caused 50% or more inhibition of B16 cell tumor growth (FIG.22).

b) Human colorectal cancer-derived COLO205 cells

OP10-9 and OP10-11 caused about 70% or more inhibition of COLO205 celltumor growth (FIG. 23).

c) Human lung cancer-derived A549 cells

OP10-9, Op10-11 and OP10-12 inhibited A549 cell tumor growth by about50% to 80% in tumor volume and about 35% to 70% in tumor weight (FIG.24).

Moreover, the r9-OP10-11 oligopeptide was also studied for its in vivoanti-tumor growth effect.

1.0×10⁶ COLO205 cells were inoculated subcutaneously into nude mice toprepare tumor of COLO205 cells. At 10 days after inoculation, a solutionof r9-OP10-11 (35 nmol)/PBS was administered in a volume of 0.1 mlaround the tumor whose diameter reached 5 to 6 mm (about 100 mm³) At 7,14 and 21 days after this administration, the same administration wasrepeated. The long and short axes of tumor were measured to calculatethe tumor volume (=long axis×(short axis)²×π/6). After the mice werefinally euthanized with carbon dioxide gas, tumor in each mouse wasphotographed. Subsequently, the tumor was excised and measured for itswet weight.

As a result, r9-OP10-11 inhibited the growth of human colorectalcancer-derived COLO205 cell tumor already formed under the skin of nudemice by about 65% in tumor volume and about 60% in tumor weight (FIG.28).

This result indicated that the r9-OP10-11 oligopeptide showed an in vivoanti-tumor growth effect.

In view of the foregoing, the siRNAs and GEF-1/C constituentoligopeptides of the present invention were found to have the ability toinhibit both in vitro and in vivo tumor growth through inhibition ofGEF-1 expression by the siRNAs and through inhibition of GEF-1 functionsby the GEF-1/C constituent oligopeptides.

INDUSTRIAL APPLICABILITY

The present invention is used as an anti-tumor agent having ananti-angiogenic effect and/or an anti-tumor growth effect.

SEQUENCE LISTING FREE TEXT

SEQ ID NOs: 13 to 43: synthetic nucleotides

SEQ ID NOs: 44 to 67: synthetic peptides

1-28. (canceled)
 29. A method for inhibiting tumor growth, which methodcomprising administering, to one or more cancer cells or to a mammal, aneffective amount of a polypeptide comprising at least the C region, or apart thereof, of a galactosylceramide expression factor-1 (GEF-1), saidpolypeptide excluding the Q region of the GEF-1.
 30. The methodaccording to claim 29, wherein the polypeptide is: (a) a polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO:8, 10 or12; or (b) a polypeptide having an anti-tumor growth effect andconsisting of an amino acid sequence having a homology of about 85% ormore with the amino acid sequence set forth in SEQ ID NO:8, 10 or 12.31. The method according to claim 29, wherein the polypeptide is: (a) isa polypeptide comprising at least ten consecutive amino acid residuesfrom the sequence set forth in SEQ ID NO:8, 10 or 12; or (b) is apolypeptide having an anti-tumor growth effect and consisting of anamino acid sequence having a deletion, substitution or addition of oneor more amino acids in a sequence of at least ten consecutive aminoacids from the sequence set forth in SEQ ID NO:8, 10 or
 12. 32. Apolypeptide according to claim 29, wherein the polypeptide comprises theamino acid sequence set forth in any of SEQ ID NOS:44-65.
 33. A methodfor inhibiting tumor growth, which method comprises administering, toone or more cancer cells or to a mammal, an effective amount of apolynucleotide that encodes a polypeptide comprising at least the Cregion, or a part thereof, of a galactosylceramide expression factor-1(GEF-1), said polypeptide excluding the Q region of the GEF-1.
 34. Themethod according to claim 33, wherein the polynucleotide encodes: (a) apolypeptide consisting of the amino acid sequence set forth in SEQ IDNO:8, 10 or 12; or (b) a polypeptide having an anti-tumor growth effectand consisting of an amino acid sequence having a homology of about 85%or more with the amino acid sequence set forth in SEQ ID NO:8, 10 or 12.35. The method according to claim 33, wherein the polynucleotideencodes: (a) a polypeptide comprising at least ten consecutive aminoacid residues from the sequence set forth in SEQ ID NO:8, 10 or 12; or(b) a polypeptide having an anti-tumor growth effect and consisting ofan amino acid sequence having a deletion, substitution or addition ofone or several amino acids in a sequence of at least ten consecutiveamino acids from the sequence set forth in SEQ ID NO:8, 10 or
 12. 36.The method according to claim 33, wherein the polynucleotide is: (a) apolynucleotide consisting of the nucleotide sequence set forth in SEQ IDNO:7, 9 or 11; or (b) a polynucleotide that encodes a polypeptide havingan anti-tumor growth effect and is hybridizable, under stringentconditions, with the complement of a polynucleotide consisting of thenucleotide sequence set forth in SEQ ID NO:7, 9 or
 11. 37. A method forinhibiting tumor growth, which method comprises administering, to one ormore cancer cells or to a mammal, an effective amount of an expressioninhibitor of a galactosylceramide expression factor-1 (GEF-1).
 38. Themethod according to claim 37, wherein the expression inhibitor of theGEF-1 is an siRNA or an shRNA against a polynucleotide encoding theGEF-1.
 39. The method according to claim 38, wherein the polynucleotideencoding the GEF-1 comprises a nucleotide sequence that encodes: (a) apolypeptide consisting of the amino acid sequence set forth in SEQ IDNO:2, 4 or 6; or (b) a polypeptide having a tumor growth effect andconsisting of an amino acid sequence having a homology of about 85% ormore with the amino acid sequence set forth in SEQ ID NO:2, 4 or 6; 40.The method according to claim 38, wherein the polynucleotide encodingthe GEF-1 is: (a) a polynucleotide consisting of the nucleotide sequenceset forth in SEQ ID NO:1, 3 or 5; or (b) a polynucleotide that encodes apolypeptide having a tumor growth effect and is hybridizable, understringent conditions, with the complement of a polynucleotide consistingof a nucleotide sequence set forth in SEQ ID NO:1, 3 or
 5. 41. A methodfor inhibiting angiogenesis, which method comprises administering, toone or more cancel cells or to a mammal, an effective amount of apolypeptide comprising at least the C region, or a part thereof, ofgalactosylceramide expression factor-1 (GEF-1), said polypeptideexcluding the Q region of the GEF-1.
 42. The method according to claim41, wherein the polypeptide is: (a) a polypeptide consisting of theamino acid sequence set forth in SEQ ID NO: 8, 10 or 12; or (b) apolypeptide having an anti-angiogenic effect and consisting of an aminoacid sequence having a homology of about 85% or more with the amino acidsequence set forth in SEQ ID NO: 8, 10 or
 12. 43. The method accordingto claim 41, wherein the polypeptide is: (a) a polypeptide comprising atleast ten consecutive amino acid residues from the sequence set forth inSEQ ID NO: 8, 10 or 12; or (b) a polypeptide having an anti-angiogeniceffect and consisting of an amino acid sequence having a deletion,substitution or addition of one or several amino acids in a sequence ofat least ten consecutive amino acids from the sequence set forth in SEQID NO: 8, 10 or
 12. 44. The method according to claim 43, wherein thepolypeptide comprises the amino acid sequence set forth in any of SEQ IDNOS: 44-65.
 45. A method for inhibiting angiogenesis, which methodcomprises administering, to one or more cancer cells or a mammal, aneffective amount of a polynucleotide that encodes a polypeptidecomprising at least the C region, or a part thereof, ofgalactosylceramide expression factor-1 (GEF-1), said polypeptideexcluding the Q region of the GEF-1.
 46. The method according to claim45, wherein the polynucleotide encodes: (a) a polypeptide consisting ofthe amino acid sequence set forth in SEQ ID NO: 8, 10 or 12; or (b) apolypeptide having an anti-angiogenic effect and consisting of an aminoacid sequence having a homology of about 85% or more with the amino acidsequence set forth in SEQ ID NO: 8, 10 or
 12. 47. The method accordingto claim 45, wherein the polynucleotide encodes: (a) a polypeptidecomprising at least ten consecutive amino acid residues from thesequence set forth in SEQ ID NO: 8, 10 or 12; or (b) a polypeptidehaving an anti-angiogenic effect and consisting of an amino acidsequence having deletion, substitution or addition of one or severalamino acids in a sequence of at least ten consecutive amino acids fromthe sequence set forth in SEQ ID NO: 8, 10 or
 12. 48. The methodaccording to claim 45, wherein the polynucleotide is: (a) apolynucleotide consisting of the nucleotide sequence set forth in SEQ IDNO: 7, 9 or 11; or (b) a polynucleotide that encodes a polypeptidehaving an anti-angiogenic effect and is hybridizable, under stringentconditions, with the complement of a polynucleotide consisting of thenucleotide sequence set forth in SEQ ID NO: 7, 9 or
 11. 49. A method forinhibiting angiogenesis, which method comprises administering, to one ormore cancer cells or a mammal, an effective amount of an expressioninhibitor of a galactosylceramide expression factor-1 (GEF-1).
 50. Themethod according to claim 49, wherein the expression inhibitor of theGEF-1 is an siRNA or an shRNA against a polynucleotide encoding theGEF-1.
 51. The method according to claim 50, wherein the polynucleotideencoding the GEF-1 comprises a nucleotide sequence that encodes: (a) apolypeptide consisting of the amino acid sequence set forth in SEQ IDNO: 2, 4 or 6; or (b) a polypeptide having an angiogenic effect andconsisting of an amino acid sequence having a homology of about 85% ormore with the amino acid sequence set forth in SEQ ID NO: 2, 4 or
 6. 52.The method according to claim 50, wherein the polynucleotide encodingthe GEF-1 is: (a) a polynucleotide consisting of the nucleotide sequenceset forth in SEQ ID NO: 1, 3 or 5; or (b) a polynucleotide that encodesa polypeptide having an angiogenic effect and is hybridizable, understringent conditions, with the complement of a polynucleotide consistingof a nucleotide sequence set forth in SEQ ID NO: 1, 3 or
 5. 53. Apolypeptide consisting of at least ten consecutive amino acid residuesfrom the sequence set forth in SEQ ID NO: 8, 10 or 12, said polypeptidehaving an anti-tumor growth effect or an anti-angiogenic effect.
 54. Thepolypeptide according to claim 53, which: (a) consists of the amino acidsequence set forth in any of SEQ ID NOS: 44-65; or (b) has an anti-tumorgrowth effect or an anti-angiogenic effect and consists of an amino acidsequence having deletion, substitution or addition of one or severalamino acids from the amino acid sequence set forth in any of SEQ ID NOS:44-65.